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Melatonin and taurine reduce early glomerulopathy in diabetic rats
Free Radical Biology & Medicine, Vol. 26, Nos. 7/8, pp. 944 –950, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter
PII S0891-5849(98)00276-7
Original Contribution MELATONIN AND TAURINE REDUCE EARLY GLOMERULOPATHY IN DIABETIC RATS HUNJOO HA, MI-RA YU,
and
KYUNG HWAN KIM
Department of Pharmacology, Yonsei University College of Medicine, Seoul, Korea (Received 13 July 1998; Revised 29 September 1998; Accepted 29 September 1998)
Abstract—Oxidative stress occurs in diabetic patients and experimental models of diabetes. We examined whether two antioxidants, melatonin and taurine, can ameliorate diabetic nephropathy. Enhanced expression of glomerular TGF-b1 and fibronectin mRNAs and proteinuria were employed as indices of diabetic nephropathy. Experimental diabetes was induced by intravenous injection of streptozotocin 50 mg/kg. Two days after streptozotocin, diabetic rats were assigned to one of the following groups: i) untreated; ii) melatonin supplement by 0.02% in drinking water; or iii) taurine supplement by 1% in drinking water. Four weeks after streptozotocin, diabetic rats (n 5 6: plasma glucose 516 6 12 mg/dl) exhibited 6.1 fold increase in urinary protein excretion, 1.4 fold increase in glomerular TGF-b1 mRNA, 1.7 fold increase in glomerular fibronectin mRNA, 2.2 fold increase in plasma lipid peroxides (LPO), and 44 fold increase in urinary LPO excretion above the values in control rats (n 5 6: plasma glucose 188 6 14 mg/dl). Chronic administration of melatonin (n 5 6) and taurine (n 5 6) prevented increases in glomerular TGF-b1 and fibronectin mRNAs and proteinuria without having effect on blood glucose. Both treatments reduced lipid peroxidation by nearly 50%. The present data demonstrate beneficial effects of melatonin and taurine on early changes in diabetic kidney and suggest that diabetic nephropathy associated with hyperglycemia is largely mediated by oxidative stress. Science © 1999 Elsevier Science Inc. Keywords—Diabetic glomerulopathy, Oxidative stress, Melatonin, Taurine, TGF-b1, Fibronectin, Streptozotocin, Free radical
INTRODUCTION
increased in the kidneys of experimental diabetic rats [6,7,12]. In addition, high glucose directly increased hydrogen peroxide production by murine mesangial cells [15] and lipid peroxidation of glomeruli [8] and glomerular mesangial cells [9 –11]. Moreover, high glucoseinduced extracellular matrix protein synthesis, an accepted in vitro model of diabetic nephropathy [18,19], were effectively attenuated by antioxidants [9 –11]. Several potential beneficial actions of antioxidant administration on diabetic neuropathy and vascular dysfunction have been reported in human and experimental diabetes [20 –22]. The present study was conducted to examine whether two antioxidants, melatonin and taurine, could ameliorate pathophysiology of diabetic nephropathy, such as enhanced expression of glomerular TGF-b1 and fibronectin mRNAs and proteinuria in streptozotocininduced diabetic rats. Melatonin, the chief secretory product of the pineal gland, was recently found to provide indirect as well as direct protection against free radical attack because it
Hyperglycemia, the main determinant of initiation [1,2] and progression [2] of diabetic microvascular complications including nephropathy, would not only generate more reactive oxygen metabolites but also attenuate antioxidative mechanisms through nonenzymatic glycosylation of antioxidative enzymes. Therefore, oxidative stress has been considered to be a common pathogenetic factor of diabetic nephropathy [3] as in other complications [4,5]. Although many studies from this laboratory [6 –9] and those of others [10 –15] have supported this hypothesis, studies of the effects of antioxidants on diabetic kidney are limited and results have been inconsistent [12–14,16,17]. Two indices of oxidative stress, lipid peroxides (LPO) and 8-hydroxydeoxyguanosine, were Address correspondence to: Hunjoo Ha, Department of Pharmacology, Yonsei University College of Medicine, Seoul 120-752, Korea; Tel: (82) 2-2-361-5233; Fax: (82) 2-313-1894; E-Mail: [email protected] 944
Antioxidants and diabetic glomerulopathy
stimulates antioxidative enzymes besides its direct scavenging ability [23]. As reviewed by Reiter et al. [23], mealtonin protects various oxidative tissue injury in vivo as well as in vitro. Taurine (2-aminoethane sulfonic acid), an abundant free amino acid in the cytosol of all organs including the kidney, is an antioxidant agent [24,25] and attenuated oxidative glomerular injury [12, 26]. Accumulation of mesangial cell extracellular matrix proteins such as collagen, laminin, and fibronectin leads to mesangial expansion which is most closely associated with loss of renal function in diabetes mellitus [27]. The mRNA levels for extracellular matrix proteins increased at early stage of experimental diabetes [28 –30]. Expression of transforming growth factor (TGF)-b, the final common mediator of the principal lesions of diabetic nephropathy such as renal hypertrophy and mesangial expansion [31,32], also increased during the early phase of diabetic nephropathy [29,30,33–38].
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whose urine glucose concentrations .2,000 mg/dl were assigned to one of the following groups: i) untreated; ii) melatonin supplement by 0.02% melatonin in drinking water; and iii) taurine supplement by 1% taurine in drinking water for 4 wk. Preliminary experiments were conducted to determine effective dosage of melatonin and taurine to attenuate proteinuria of diabetic rats based on the previous reports [12,23,26].
Measurements of plasma glucose and urinary protein excretion Blood samples were obtained from tail veins. Urine was collected for 24 h. Plasma glucose was measured by the glucose oxidase method using a commercial diagnostic kit (510-DA; Sigma Chemical Co.). Urinary protein was measured by means of a quantitative reaction with Coomassie blue using a Bio-Rad protein assay (500 – 0006; Hercules, CA).
MATERIALS AND METHODS
Male, 7-wk-old, Sprague-Dawley rats were obtained from the animal facility of the Yonsei University College of Medicine. Four experimental groups were formed: streptozotocin-induced diabetic rats (DR; n 5 6), agematched control rats (CR; n 5 6), diabetic rats treated with melatonin (DR1M; n 5 6), and diabetic rats treated with taurine (DR1T; n 5 6). All rats were maintained on standard rat chow (Samyang rat chow, Seoul, Korea) and tap water ad lib. Streptozotocin was obtained from the Sigma Chemical Company (St. Louis, MO). Deoxycytidine-59-[a-32P] triphosphate (sp. act. 5 3000 Ci/mmol) and nylon membrane were obtained from the Amersham Corporation (Arlington Heights, IL). cDNA probes for TGF-b1 and fibronectin were purchased from the American Type Culture Collection (Rockville, MD). Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was constructed based on the previously published sequence using the polymerase chain reaction (PCR) [39]. All other chemicals used were of analytical grade.
Induction of experimental diabetes mellitus Diabetes was induced by the intravenous injection of 50 mg/kg of streptozotocin (dissolved in pH 4.5 citrate buffer immediately before injection) into the rat tail vein. Control rats received the same volume of citrate buffer (2.5 ml/kg). Induction of the diabetic state was confirmed by estimating urine glucose with commercial enzymatic test strips (Bayer-Sankyo Co., Ltd.; Tokyo, Japan) at two days after the injection of streptozotocin. Diabetic rats
Measurement of LPO A modification of the thiobarbituric acid method of Ohkawa et al. [40] was used to measure the level of LPO using tetraethoxypropane as a standard [6,8,9]. Aliquots of plasma and urine (80 ml) were mixed with 80 ml 8% SDS and a reaction mixture consisting of 120 ml of 0.8% 2-thiobarbituric acid and 120 ml 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 g for 5 min to precipitate interfering particulate materials. The amount of LPO formed was measured by spectrofluorometry (SPF-500C; SLM Instruments, Inc; Urbana, IL) at an emission wavelength of 553 nm with an excitation wavelength of 515 nm (Fig. 1).
Northern blot analysis Total RNA was isolated from the isolated glomeruli using the method of Chomczynski and Sacchi [41]. In brief, glomeruli were isolated using a standard sieving method and homogenized with 4 M guanidium thiocyanate. Total protein and DNA were extracted with acid phenol, and RNA precipitated with isopropanol. After washing with ethanol, the samples were dried under vacuum centrifugation and the amount of RNA quantitated by measuring the absorbance at 260 nm using a spectrophotometer. Twenty mg of total RNA was electrophoresed through a 1.2% agarose gel with 2.2 M formaldehyde. The RNA from the gel was transferred onto nylon membranes using a capillary transfer and
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Fig. 1. Effect of melatonin and taurine on urinary LPO excretion and plasma LPO of streptozotocin induced diabetic rats. Values are mean SE obtained from 6 rats/group 4 wk after treatment. CR5 control rats, DR5 diabetic rats, DR1M5 diabetic rats treated with melatonin, DR1T5 diabetic rats treated with taurine. *p , .05 compared to CR.
covalently cross-linked to the membrane with UV light using a gene-linker (Bio-Rad Corp.). Prehybridization was performed for 5 h at 42°C using the GIBCO BRL prehybridization buffer (15592-017; Geithersburg, MD). Hybridization was conducted for 20 h at 42°C using excised cDNA inserts as probes after [32P]dCTP-labeling by a random primer extension method (Pharmacia Biotech; Uppsala, Sweden). The membranes were washed three times for 30 min: first in 23 sodium chloride and sodium citrate (SSC; 13 SSC is 0.15 M NaCl and 0.015 M sodium citrate, adjusted to pH 7.0) with 0.1% sodium dodecyl sulfate at room temperature; second in 0.23 SSC with 0.1% SDS at room temperature; and third in 0.23 SSC with 0.1% SDS at 55°C. Autoradiography was performed by exposing the blots to Kodak X-Omat K XK-1 X-ray film with intensifying screens at 270°C for three to seven days for fibronectin, one to three days for TGF-b1, or several hr for GAPDH. The blots were then rehybridized with a 32 P-labeled human GAPDH cDNA probe as an internal control to assess RNA quantity and integrity. Quantitation of mRNA signals was performed by densitometry using a Bio-Rad Imaging Densitometer Model GS-610 and normalized with GAPDH mRNA signals. Analysis of data All results are expressed as means standard error (SE). Analysis of variance was used to assess the differences between multiple groups. If the F statistic was significant, the mean values obtained from each group were then compared by Scheffe’s F test. A p value , .05 was used as the criterion for a statistically significant difference.
RESULTS
Chronic effects of melatonin and taurine on body weight, proteinuria, and lipid peroxidation of diabetic rats Rats with urine glucose concentrations .2,000 mg/dl at two days after streptozotocin injection were used for the study. Diabetic rats failed to gain body weight compared to control rats and exhibited polyuria at 4 wk after injection of streptozotocin. But diabetic rats were not ketotic or acidotic (data not shown). Neither melatonin nor taurine significantly affect body weight of diabetic rats, although diabetic rats treated with melatonin weighed 14% less than the untreated and the taurine treated diabetic rats. No visible side effects of melatonin were noted during the present study. Neither melatonin nor taurine caused any discernible effects on plasma glucose levels of diabetic rats. The mean urinary protein excretion in diabetic rats was 6.1 fold greater than that in control rats. Both melatonin and taurine effectively prevented diabetic proteinuria. The data are summarized in Table 1. As summarized in Fig. 1, urinary LPO excretion was 44 fold higher in diabetic rats compared to control rats. This increase in urinary LPO was partly due to the increase in plasma LPO (2.2 fold) in diabetic rats. Since preliminary study showed no difference in serum levels of cholesterol and triglyceride between diabetic and control rats, plasma LPO was expressed as unit per volume. Both melatonin and taurine reduced lipid peroxidation associated with diabetes by nearly 50%, although the differences between the melatonin and taurine treated diabetic rats and the untreated diabetic rats did not reach statistical significance.
Antioxidants and diabetic glomerulopathy Table 1. Effects of Melatonin and Taurine on Body Weight, Plasma Glucose, and Urine Volume of Streptozotocin-Induced Diabetic Rats
Body weight, g Plasma glucose, mg/dl Urine volume, ml/24 h Urinary protein Excretion, mg/24 h
CR
DR
DR 1 M
DR 1 T
275 6 6 188 6 14
207 6 14* 516 6 12*
178 6 21* 490 6 54*
207 6 17* 477 1 11*
861
101 6 23*
62 6 16
61 6 18
20 6 2
121 6 26*
38 6 51
39 6 71
Values are mean 6SE obtained from 6 rats/group at 4 weeks after treatment (now 11- to 12-wk-old rats). CR 5 control rats, DR 5 diabetic rats, DR 1 M 5 diabetic rats treated with melatonin, DR 1 T 5 diabetic rats treated with taurine. *P , 0.05 compared to CR. 1 P , 0.05 compared to DR.
Relationship among plasma glucose, urinary protein and LPO excretions, and plasma LPO When the data obtained from all the groups in the present study were pooled and analyzed, the correlation coefficient value always exhibited a very significant difference between any of two variables among plasma glucose, urinary protein and LPO excretions, and plasma LPO. Table 2 summarizes the correlation coefficient values after excluding the data from control rats. Urinary
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Table 2. Correlation Coefficients among Plasma Glucose, Urinary Protein and Lipid Peroxides (LPO) Excretions, and Plasma LPO
Urinary Protein vs Plasma Glucose Urinary Protein vs Plasma LPO Urinary Protein vs Urinary LPO Urinary LPO vs Plasma Glucose Urinary LPO vs Plasma LPO Plasma LPO vs Plasma Glucose
R2
P
0.107 0.504 0.436 0.376 0.357 0.340
0.1857 0.0014 0.0029 0.0068 0.0113 0.0139
Data obtained from 6 streptozotocin-induced diabetic rats, 6 melatonin treated diabetic rats, and 6 taurine treated diabetic rats 4 weeks after treatment. LPO 5 lipid peroxides.
protein excretion had no correlation with plasma glucose. The relationship between urinary protein excretion and plasma LPO was the most significant followed by the relationship between urinary excretion of protein and LPO. Effects of melatonin and taurine on glomerular expression of TGF-b1 and fibronectin mRNAs in diabetic rats As shown in Fig. 2, both TGF-b1 and fibronectin mRNA levels significantly increased in the glomeruli of
Fig. 2. Effect of melatonin and taurine on glomerular expression of TGF-b1 and fibronectin mRNAs in streptozotocin induced diabetic rats. Northern blot analysis was done as described in Materials and Methods. Values are mean 6 SE obtained from 6 rats/group 4 wk after treatment. CR5 control rats, DR5 diabetic rats, DR1M5 diabetic rats treated with melatonin, DR1T5 diabetic rats treated with taurine. *p , .05 compared to CR. 1p , .05 compared to DR.
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diabetic rats 4 wk after the induction of diabetes. There was a 1.4 fold increase in TGF-b1 mRNA and 1.7 fold increase in fibronectin mRNA expression in untreated diabetic rats over control rats. Melatonin and taurine effectively prevented the increases in TGF-b1 and fibronectin mRNA levels. mRNA expressions of both TGF-b1 and fibronectin of diabetic rats treated with melatonin were statistically lower than those of control rats. DISCUSSION
Excessive deposition of extracellular matrix proteins and subsequent mesangial expansion is the principal structural lesion of diabetic kidney [27]. Increased expression of mRNA encoding for TGF-b1 [29,30,33–38] and extracellular matrix proteins [28 –30] occurs prior to mesangial expansion. Thus, understanding the mechanisms involved in these early altered expression of TGF-b1 and extracellular matrix protein genes is necessary to find ways of preventing diabetic glomerular injury and preserve renal function. At 4 wk after the injection of streptozotocin, rats had typical characteristics of diabetes mellitus, such as hyperglycemia, polyuria, and growth retardation. These diabetic rats also exhibited increase in urinary protein excretion but not selective mesangial expansion (data not shown). Glomerular TGF-b1 mRNA expression of diabetic rats 4 wk after injection increased 1.4 fold above control which generally agrees with previous studies [29,33–38]. Increased glomerular TGF-b1 mRNA expression was demonstrated at 2 to 3 days after streptozotocin by either in situ hybridization [29,36] or PCR analysis [35] and 2– 6 wk after streptozotocin by Northern blot analysis [33, 34,37,38]. These differences attribute to both the method of mRNA detection and different stage of diabetic renal injury during each study. The glomerular extracellular matrix is composed of type IV, V, and VI collagen, heparan sulfate proteoglycan, laminin, and fibronectin. In situ immuno-histochemisty [29] demonstrated marked increase in expression of glomerular a1(IV) collagen mRNA in diabetic rats as early as 3 days after streptozotocin. Utilizing Northern blot analysis, Fukui et al. [28] reported increased mRNA levels for a1(IV) collagen, laminin B1 and B2, and a1(I) and a1(III) collagen in diabetic rats 4 wk after streptozotocin before morphological thickening of basement membrane occurred. Progressive increase in fibronectin and type IV collagen mRNA expression was observed in the glomeruli of diabetic rats from 4 to 12 months after streptozotocin [30]. The present study demonstrates significant increase in mRNA encoding fibronectin as early as 4 wk after the onset of diabetes.
Tight glycemic control ameliorated increased glomerular TGF-b1 [29,33–35,37,38] and extracellular matrix protein [28,29] mRNA expression suggesting that hyperglycemia is the main determinant of TGF-b1 and extracellular matrix protein synthesis in diabetic kidney and that whatever mechanisms involved in this alteration is due to diabetic condition but not the direct effect of streptozotocin per se. The specific mechanisms involved in modulating expression of TGF-b1 and extracellular matrix protein genes by high glucose are not completely understood. However, the causal relationship between oxidative stress and diabetic nephropathy has been demonstrated by the fact that indices of oxidative stress is increased in the diabetic kidney [6,7,12]; that oxidative stress associated with high glucose has a relevant effect on target cells in vitro [8 –11]; and that inhibition of oxidative stress in vitro masks the manifestations of the disease [9 –11]. Yet the previous studies of the influence of antioxidants on diabetic kidney have yielded inconsistent result. Vitamin E ameliorated increase in glomerular filtration rate after 2 wk of diabetes, reduced albuminuria at 10 wk [13], and prevented increase in glomerular TGF-b1 protein at 2 months after streptozotocin [14]. By contrast, we did not found any protective effect of vitamin E on albuminuria of streptozotocin-induced diabetic rats [17]. Trachtman and coworkers [12] reported greater decline in renal function and accelerated glomerulosclerosis in diabetic rats treated with vitamin E. In the same study [12], dietary supplement with taurine 1% in drinking water for 1 year attenuated proteinuria and glomerulosclerosis in diabetic rats. Present study also demonstrates effective prevention of proteinuria by taurine at dose inhibiting lipid peroxidation. In addition, taurine totally abolished increases in glomerular TGF-b1 and fibronectin mRNA expression, supporting the previous long-term beneficial effect of taurine on diabetic glemerulosclerosis [12]. In spite of potent antioxidant effect of melatonin on brain, lung, and liver [22], no study has been conducted to investigate protective effect of melatonin on the kidney from oxidative damage. Melatonin at dose inhibiting lipid peroxidation prevented increased glomerular TGF-b1 and fibronectin mRNA expresssion and proteinuria, suggesting that beneficial effect of melatonin may result from its antioxidative property. A recent study [42] demonstrating that chronic prooxidant state increased renal TGF-b1 and extracellular matrix protein mRNA expression in the rats supports our results. In addition, high glucose produced more hydrogen peroxide by murine mesangial cells [15] and hydrogen peroxide increased TGF-b1 and fibronectin synthesis in mesangial cells [43]. The significance of lowered TGF-b1 and fibronectin mRNAs in the glomeruli of diabetic rats treated with melatonin below control re-
Antioxidants and diabetic glomerulopathy
mains to be defined in future studies investigating the long-term effects of melatonin on control as well as diabetic kidneys. Neither melatonin nor taurine has any effect on plasma glucose. Lack of correlation between proteinuria and plasma glucose but strong correlation between proteinuria and lipid peroxidation further suggest that oxidative stress associated with hyperglycemia has a role on increased expression of glomerular TGF-b1 and fibronectin mRNAs and proteinuria of diabetic rats. Abolishment of glomerular TGF-b1 and fibronectin mRNA increments and proteinuria in diabetic rats with 50% reduction of lipid peroxidation by melatonin and taurine suggest that oxidative stress above threshold is a pathogenic factor of diabetic nephropathy. The ability of antioxidants to prevent increases in glomerular TGF-b1 and fibronectin mRNAs in diabetic kidney is consistent with previous in vitro studies which demonstrated effective inhibition of high glucose-induced TGF-b1 and extracellular matrix protein synthesis [9 –11,44]. Activation of PKC, one of the major biochemical pathway inducing diabetic nephropathy [45], is sensitively regulated through redox changes in sulfhydryl groups of cystein-rich regions in PKC [46,47]. Antioxidants including vitamin E and taurine suppressed increases in PKC and TGF-b synthesis by mesangial cells in response to high glucose [44], suggesting antioxidants reduce the level of cellular oxidants and protect reactive protein sulfhydryls from oxidation. Vitamin E, but not taurine, may have direct effects on interaction of the PKC system with diacyl glycerol (DAG) , since it directly increase DAG kinase leading to inhibition of PKC activation [14,44]. On the other hand, our preliminary study [43] demonstrated that inhibition of PKC effectively blocked phorbol ester-, high glucose-, and H2O2-induced TGF-b1 and fibronectin synthesis by mesangial cells [43] and suggested modulatory role of PKC on oxidative glomerular injury in diabetes. Thus, coordinate interaction between oxidative stress and PKC under hyperglycemia may lead to increase in glomerular TGF-b1 and fibronectin mRNAs. In conclusion, our data demonstrate beneficial effects of melatonin and taurine on early changes in diabetic kidney and suggest that diabetic nephropathy associated with hyperglycemia is largely mediated by oxidative stress. Acknowledgements — We thank Professor Hi Bahl Lee at Hyonam Kidney Laboratory, Soon Chun Hyang University, for his continued interest in our work and constructive criticism and Dr. Sung Il Kim for providing cDNA probes for TGF-b1 and GAPDH. This work was supported in part by a grant from Korea Science and Engineering foundation (KOSEF 94-0403-07-01-3). Portions of this work were presented at the 30th Annual Meeting of American Society of Nephrology (San Antonio, TX, USA, 1997) and appeared in abstract form.
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[37] Young, B. A.; Johnson, R. J.; Alpers, C. E.; Eudora, E.; Gordon, K.; Gloege, J.; Couseer, W.G. Cellular events in the evolution of experimental diabetic nephropathy. Kidney Int. 47:935–944; 1995 [38] Han, D. C.; Kim, Y. J.; Cha, M. K.; Song, K. I.; Kim, J. H.; Lee, E. Y.; Ha, H.; Lee, H.B. Glucose control suppressed the glomerular expression of TGF-b1 and the progression of experimental diabetic nephropathy. J. Am. Soc. Nephrol. 7:1870 abstr. (1996). [39] Piechaczyk, M.; Blanchard, J. M.; Marty, L.; Dani. Ch.; Panabieres, F.; El Sabouty, S.; Fort, Ph.; Jeanteur, Ph. Post-transcriptional regulation of glyceraldehyde-3- phosphate-dehydrogenase gene expression in rat tissues. Nucleic Acid Res. 12:6951– 6963; 1984. [40] Ohkawa, H.; Ohishi, N.; Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95:351–358; 1979. [41] Chomczynski, P.; Sacchi, N. Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156 –159; 1987. [42] Nath, K. A.; Grande, J.; Croatt, A.; Haugen, J.; Kim, Y.; Rosenberg, M. E. Redox regulation of renal DNA synthesis, transforming growth factor-b1 and collagen gene expression. Kidney Int. 53:367–381, 1998. [43] Ha, H.; Kim, K. H. Role of protein kinase C-mediated oxidative stress in increased TGF-b1 and fibronectin mRNAs in mesangial cells: Implications for diabetic nephropathy. Nephrology 3(Suppl 1):A736 abstr. (1997). [44] Studer, R. K.; Craven, P. A.; DeRubertis, F. R. Antioxidant inhibition of protein kinase C-signaled increases in transforming growth factor-beta in mesangial cells. Met. Clin. Exp. 46:918 – 925; 1997. [45] King, G. L.; Ishii, H.; Koya, D. Diabetic vascular dysfunctions: A model of excessive activation of protein kinase C. Kidney Int. 52(Suppl 60):S77–S85; 1997. [46] Gopalkrishna, R.; Anderson, W. B. Reversible oxidative activation and inactivation of protein kinase C by the mitogen/tumor promotor periodate. Arch. Biochem. Biophys. 285:382–387; 1991. [47] Gschwendt, M.; Kittstein, W.; Marks, F. Protein kinase C activation by phorbol esters: Do cysteine rich regions and pseudosubstrate motifs play a role? Trends Biochem. Sci. 16:167–169; 1991.
ABBREVIATIONS
CR— control rats DR— diabetic rats GAPDH— glyceraldehyde-3-phosphate dehydrogenase LPO—lipid peroxides M—melatonin PCR—polymerase chain reaction PKC—protein kinase C T—taurine TGF-b1—transforming growth factor-b1
PII S0891-5849(98)00276-7
Original Contribution MELATONIN AND TAURINE REDUCE EARLY GLOMERULOPATHY IN DIABETIC RATS HUNJOO HA, MI-RA YU,
and
KYUNG HWAN KIM
Department of Pharmacology, Yonsei University College of Medicine, Seoul, Korea (Received 13 July 1998; Revised 29 September 1998; Accepted 29 September 1998)
Abstract—Oxidative stress occurs in diabetic patients and experimental models of diabetes. We examined whether two antioxidants, melatonin and taurine, can ameliorate diabetic nephropathy. Enhanced expression of glomerular TGF-b1 and fibronectin mRNAs and proteinuria were employed as indices of diabetic nephropathy. Experimental diabetes was induced by intravenous injection of streptozotocin 50 mg/kg. Two days after streptozotocin, diabetic rats were assigned to one of the following groups: i) untreated; ii) melatonin supplement by 0.02% in drinking water; or iii) taurine supplement by 1% in drinking water. Four weeks after streptozotocin, diabetic rats (n 5 6: plasma glucose 516 6 12 mg/dl) exhibited 6.1 fold increase in urinary protein excretion, 1.4 fold increase in glomerular TGF-b1 mRNA, 1.7 fold increase in glomerular fibronectin mRNA, 2.2 fold increase in plasma lipid peroxides (LPO), and 44 fold increase in urinary LPO excretion above the values in control rats (n 5 6: plasma glucose 188 6 14 mg/dl). Chronic administration of melatonin (n 5 6) and taurine (n 5 6) prevented increases in glomerular TGF-b1 and fibronectin mRNAs and proteinuria without having effect on blood glucose. Both treatments reduced lipid peroxidation by nearly 50%. The present data demonstrate beneficial effects of melatonin and taurine on early changes in diabetic kidney and suggest that diabetic nephropathy associated with hyperglycemia is largely mediated by oxidative stress. Science © 1999 Elsevier Science Inc. Keywords—Diabetic glomerulopathy, Oxidative stress, Melatonin, Taurine, TGF-b1, Fibronectin, Streptozotocin, Free radical
INTRODUCTION
increased in the kidneys of experimental diabetic rats [6,7,12]. In addition, high glucose directly increased hydrogen peroxide production by murine mesangial cells [15] and lipid peroxidation of glomeruli [8] and glomerular mesangial cells [9 –11]. Moreover, high glucoseinduced extracellular matrix protein synthesis, an accepted in vitro model of diabetic nephropathy [18,19], were effectively attenuated by antioxidants [9 –11]. Several potential beneficial actions of antioxidant administration on diabetic neuropathy and vascular dysfunction have been reported in human and experimental diabetes [20 –22]. The present study was conducted to examine whether two antioxidants, melatonin and taurine, could ameliorate pathophysiology of diabetic nephropathy, such as enhanced expression of glomerular TGF-b1 and fibronectin mRNAs and proteinuria in streptozotocininduced diabetic rats. Melatonin, the chief secretory product of the pineal gland, was recently found to provide indirect as well as direct protection against free radical attack because it
Hyperglycemia, the main determinant of initiation [1,2] and progression [2] of diabetic microvascular complications including nephropathy, would not only generate more reactive oxygen metabolites but also attenuate antioxidative mechanisms through nonenzymatic glycosylation of antioxidative enzymes. Therefore, oxidative stress has been considered to be a common pathogenetic factor of diabetic nephropathy [3] as in other complications [4,5]. Although many studies from this laboratory [6 –9] and those of others [10 –15] have supported this hypothesis, studies of the effects of antioxidants on diabetic kidney are limited and results have been inconsistent [12–14,16,17]. Two indices of oxidative stress, lipid peroxides (LPO) and 8-hydroxydeoxyguanosine, were Address correspondence to: Hunjoo Ha, Department of Pharmacology, Yonsei University College of Medicine, Seoul 120-752, Korea; Tel: (82) 2-2-361-5233; Fax: (82) 2-313-1894; E-Mail: [email protected] 944
Antioxidants and diabetic glomerulopathy
stimulates antioxidative enzymes besides its direct scavenging ability [23]. As reviewed by Reiter et al. [23], mealtonin protects various oxidative tissue injury in vivo as well as in vitro. Taurine (2-aminoethane sulfonic acid), an abundant free amino acid in the cytosol of all organs including the kidney, is an antioxidant agent [24,25] and attenuated oxidative glomerular injury [12, 26]. Accumulation of mesangial cell extracellular matrix proteins such as collagen, laminin, and fibronectin leads to mesangial expansion which is most closely associated with loss of renal function in diabetes mellitus [27]. The mRNA levels for extracellular matrix proteins increased at early stage of experimental diabetes [28 –30]. Expression of transforming growth factor (TGF)-b, the final common mediator of the principal lesions of diabetic nephropathy such as renal hypertrophy and mesangial expansion [31,32], also increased during the early phase of diabetic nephropathy [29,30,33–38].
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whose urine glucose concentrations .2,000 mg/dl were assigned to one of the following groups: i) untreated; ii) melatonin supplement by 0.02% melatonin in drinking water; and iii) taurine supplement by 1% taurine in drinking water for 4 wk. Preliminary experiments were conducted to determine effective dosage of melatonin and taurine to attenuate proteinuria of diabetic rats based on the previous reports [12,23,26].
Measurements of plasma glucose and urinary protein excretion Blood samples were obtained from tail veins. Urine was collected for 24 h. Plasma glucose was measured by the glucose oxidase method using a commercial diagnostic kit (510-DA; Sigma Chemical Co.). Urinary protein was measured by means of a quantitative reaction with Coomassie blue using a Bio-Rad protein assay (500 – 0006; Hercules, CA).
MATERIALS AND METHODS
Male, 7-wk-old, Sprague-Dawley rats were obtained from the animal facility of the Yonsei University College of Medicine. Four experimental groups were formed: streptozotocin-induced diabetic rats (DR; n 5 6), agematched control rats (CR; n 5 6), diabetic rats treated with melatonin (DR1M; n 5 6), and diabetic rats treated with taurine (DR1T; n 5 6). All rats were maintained on standard rat chow (Samyang rat chow, Seoul, Korea) and tap water ad lib. Streptozotocin was obtained from the Sigma Chemical Company (St. Louis, MO). Deoxycytidine-59-[a-32P] triphosphate (sp. act. 5 3000 Ci/mmol) and nylon membrane were obtained from the Amersham Corporation (Arlington Heights, IL). cDNA probes for TGF-b1 and fibronectin were purchased from the American Type Culture Collection (Rockville, MD). Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was constructed based on the previously published sequence using the polymerase chain reaction (PCR) [39]. All other chemicals used were of analytical grade.
Induction of experimental diabetes mellitus Diabetes was induced by the intravenous injection of 50 mg/kg of streptozotocin (dissolved in pH 4.5 citrate buffer immediately before injection) into the rat tail vein. Control rats received the same volume of citrate buffer (2.5 ml/kg). Induction of the diabetic state was confirmed by estimating urine glucose with commercial enzymatic test strips (Bayer-Sankyo Co., Ltd.; Tokyo, Japan) at two days after the injection of streptozotocin. Diabetic rats
Measurement of LPO A modification of the thiobarbituric acid method of Ohkawa et al. [40] was used to measure the level of LPO using tetraethoxypropane as a standard [6,8,9]. Aliquots of plasma and urine (80 ml) were mixed with 80 ml 8% SDS and a reaction mixture consisting of 120 ml of 0.8% 2-thiobarbituric acid and 120 ml 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 g for 5 min to precipitate interfering particulate materials. The amount of LPO formed was measured by spectrofluorometry (SPF-500C; SLM Instruments, Inc; Urbana, IL) at an emission wavelength of 553 nm with an excitation wavelength of 515 nm (Fig. 1).
Northern blot analysis Total RNA was isolated from the isolated glomeruli using the method of Chomczynski and Sacchi [41]. In brief, glomeruli were isolated using a standard sieving method and homogenized with 4 M guanidium thiocyanate. Total protein and DNA were extracted with acid phenol, and RNA precipitated with isopropanol. After washing with ethanol, the samples were dried under vacuum centrifugation and the amount of RNA quantitated by measuring the absorbance at 260 nm using a spectrophotometer. Twenty mg of total RNA was electrophoresed through a 1.2% agarose gel with 2.2 M formaldehyde. The RNA from the gel was transferred onto nylon membranes using a capillary transfer and
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Fig. 1. Effect of melatonin and taurine on urinary LPO excretion and plasma LPO of streptozotocin induced diabetic rats. Values are mean SE obtained from 6 rats/group 4 wk after treatment. CR5 control rats, DR5 diabetic rats, DR1M5 diabetic rats treated with melatonin, DR1T5 diabetic rats treated with taurine. *p , .05 compared to CR.
covalently cross-linked to the membrane with UV light using a gene-linker (Bio-Rad Corp.). Prehybridization was performed for 5 h at 42°C using the GIBCO BRL prehybridization buffer (15592-017; Geithersburg, MD). Hybridization was conducted for 20 h at 42°C using excised cDNA inserts as probes after [32P]dCTP-labeling by a random primer extension method (Pharmacia Biotech; Uppsala, Sweden). The membranes were washed three times for 30 min: first in 23 sodium chloride and sodium citrate (SSC; 13 SSC is 0.15 M NaCl and 0.015 M sodium citrate, adjusted to pH 7.0) with 0.1% sodium dodecyl sulfate at room temperature; second in 0.23 SSC with 0.1% SDS at room temperature; and third in 0.23 SSC with 0.1% SDS at 55°C. Autoradiography was performed by exposing the blots to Kodak X-Omat K XK-1 X-ray film with intensifying screens at 270°C for three to seven days for fibronectin, one to three days for TGF-b1, or several hr for GAPDH. The blots were then rehybridized with a 32 P-labeled human GAPDH cDNA probe as an internal control to assess RNA quantity and integrity. Quantitation of mRNA signals was performed by densitometry using a Bio-Rad Imaging Densitometer Model GS-610 and normalized with GAPDH mRNA signals. Analysis of data All results are expressed as means standard error (SE). Analysis of variance was used to assess the differences between multiple groups. If the F statistic was significant, the mean values obtained from each group were then compared by Scheffe’s F test. A p value , .05 was used as the criterion for a statistically significant difference.
RESULTS
Chronic effects of melatonin and taurine on body weight, proteinuria, and lipid peroxidation of diabetic rats Rats with urine glucose concentrations .2,000 mg/dl at two days after streptozotocin injection were used for the study. Diabetic rats failed to gain body weight compared to control rats and exhibited polyuria at 4 wk after injection of streptozotocin. But diabetic rats were not ketotic or acidotic (data not shown). Neither melatonin nor taurine significantly affect body weight of diabetic rats, although diabetic rats treated with melatonin weighed 14% less than the untreated and the taurine treated diabetic rats. No visible side effects of melatonin were noted during the present study. Neither melatonin nor taurine caused any discernible effects on plasma glucose levels of diabetic rats. The mean urinary protein excretion in diabetic rats was 6.1 fold greater than that in control rats. Both melatonin and taurine effectively prevented diabetic proteinuria. The data are summarized in Table 1. As summarized in Fig. 1, urinary LPO excretion was 44 fold higher in diabetic rats compared to control rats. This increase in urinary LPO was partly due to the increase in plasma LPO (2.2 fold) in diabetic rats. Since preliminary study showed no difference in serum levels of cholesterol and triglyceride between diabetic and control rats, plasma LPO was expressed as unit per volume. Both melatonin and taurine reduced lipid peroxidation associated with diabetes by nearly 50%, although the differences between the melatonin and taurine treated diabetic rats and the untreated diabetic rats did not reach statistical significance.
Antioxidants and diabetic glomerulopathy Table 1. Effects of Melatonin and Taurine on Body Weight, Plasma Glucose, and Urine Volume of Streptozotocin-Induced Diabetic Rats
Body weight, g Plasma glucose, mg/dl Urine volume, ml/24 h Urinary protein Excretion, mg/24 h
CR
DR
DR 1 M
DR 1 T
275 6 6 188 6 14
207 6 14* 516 6 12*
178 6 21* 490 6 54*
207 6 17* 477 1 11*
861
101 6 23*
62 6 16
61 6 18
20 6 2
121 6 26*
38 6 51
39 6 71
Values are mean 6SE obtained from 6 rats/group at 4 weeks after treatment (now 11- to 12-wk-old rats). CR 5 control rats, DR 5 diabetic rats, DR 1 M 5 diabetic rats treated with melatonin, DR 1 T 5 diabetic rats treated with taurine. *P , 0.05 compared to CR. 1 P , 0.05 compared to DR.
Relationship among plasma glucose, urinary protein and LPO excretions, and plasma LPO When the data obtained from all the groups in the present study were pooled and analyzed, the correlation coefficient value always exhibited a very significant difference between any of two variables among plasma glucose, urinary protein and LPO excretions, and plasma LPO. Table 2 summarizes the correlation coefficient values after excluding the data from control rats. Urinary
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Table 2. Correlation Coefficients among Plasma Glucose, Urinary Protein and Lipid Peroxides (LPO) Excretions, and Plasma LPO
Urinary Protein vs Plasma Glucose Urinary Protein vs Plasma LPO Urinary Protein vs Urinary LPO Urinary LPO vs Plasma Glucose Urinary LPO vs Plasma LPO Plasma LPO vs Plasma Glucose
R2
P
0.107 0.504 0.436 0.376 0.357 0.340
0.1857 0.0014 0.0029 0.0068 0.0113 0.0139
Data obtained from 6 streptozotocin-induced diabetic rats, 6 melatonin treated diabetic rats, and 6 taurine treated diabetic rats 4 weeks after treatment. LPO 5 lipid peroxides.
protein excretion had no correlation with plasma glucose. The relationship between urinary protein excretion and plasma LPO was the most significant followed by the relationship between urinary excretion of protein and LPO. Effects of melatonin and taurine on glomerular expression of TGF-b1 and fibronectin mRNAs in diabetic rats As shown in Fig. 2, both TGF-b1 and fibronectin mRNA levels significantly increased in the glomeruli of
Fig. 2. Effect of melatonin and taurine on glomerular expression of TGF-b1 and fibronectin mRNAs in streptozotocin induced diabetic rats. Northern blot analysis was done as described in Materials and Methods. Values are mean 6 SE obtained from 6 rats/group 4 wk after treatment. CR5 control rats, DR5 diabetic rats, DR1M5 diabetic rats treated with melatonin, DR1T5 diabetic rats treated with taurine. *p , .05 compared to CR. 1p , .05 compared to DR.
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diabetic rats 4 wk after the induction of diabetes. There was a 1.4 fold increase in TGF-b1 mRNA and 1.7 fold increase in fibronectin mRNA expression in untreated diabetic rats over control rats. Melatonin and taurine effectively prevented the increases in TGF-b1 and fibronectin mRNA levels. mRNA expressions of both TGF-b1 and fibronectin of diabetic rats treated with melatonin were statistically lower than those of control rats. DISCUSSION
Excessive deposition of extracellular matrix proteins and subsequent mesangial expansion is the principal structural lesion of diabetic kidney [27]. Increased expression of mRNA encoding for TGF-b1 [29,30,33–38] and extracellular matrix proteins [28 –30] occurs prior to mesangial expansion. Thus, understanding the mechanisms involved in these early altered expression of TGF-b1 and extracellular matrix protein genes is necessary to find ways of preventing diabetic glomerular injury and preserve renal function. At 4 wk after the injection of streptozotocin, rats had typical characteristics of diabetes mellitus, such as hyperglycemia, polyuria, and growth retardation. These diabetic rats also exhibited increase in urinary protein excretion but not selective mesangial expansion (data not shown). Glomerular TGF-b1 mRNA expression of diabetic rats 4 wk after injection increased 1.4 fold above control which generally agrees with previous studies [29,33–38]. Increased glomerular TGF-b1 mRNA expression was demonstrated at 2 to 3 days after streptozotocin by either in situ hybridization [29,36] or PCR analysis [35] and 2– 6 wk after streptozotocin by Northern blot analysis [33, 34,37,38]. These differences attribute to both the method of mRNA detection and different stage of diabetic renal injury during each study. The glomerular extracellular matrix is composed of type IV, V, and VI collagen, heparan sulfate proteoglycan, laminin, and fibronectin. In situ immuno-histochemisty [29] demonstrated marked increase in expression of glomerular a1(IV) collagen mRNA in diabetic rats as early as 3 days after streptozotocin. Utilizing Northern blot analysis, Fukui et al. [28] reported increased mRNA levels for a1(IV) collagen, laminin B1 and B2, and a1(I) and a1(III) collagen in diabetic rats 4 wk after streptozotocin before morphological thickening of basement membrane occurred. Progressive increase in fibronectin and type IV collagen mRNA expression was observed in the glomeruli of diabetic rats from 4 to 12 months after streptozotocin [30]. The present study demonstrates significant increase in mRNA encoding fibronectin as early as 4 wk after the onset of diabetes.
Tight glycemic control ameliorated increased glomerular TGF-b1 [29,33–35,37,38] and extracellular matrix protein [28,29] mRNA expression suggesting that hyperglycemia is the main determinant of TGF-b1 and extracellular matrix protein synthesis in diabetic kidney and that whatever mechanisms involved in this alteration is due to diabetic condition but not the direct effect of streptozotocin per se. The specific mechanisms involved in modulating expression of TGF-b1 and extracellular matrix protein genes by high glucose are not completely understood. However, the causal relationship between oxidative stress and diabetic nephropathy has been demonstrated by the fact that indices of oxidative stress is increased in the diabetic kidney [6,7,12]; that oxidative stress associated with high glucose has a relevant effect on target cells in vitro [8 –11]; and that inhibition of oxidative stress in vitro masks the manifestations of the disease [9 –11]. Yet the previous studies of the influence of antioxidants on diabetic kidney have yielded inconsistent result. Vitamin E ameliorated increase in glomerular filtration rate after 2 wk of diabetes, reduced albuminuria at 10 wk [13], and prevented increase in glomerular TGF-b1 protein at 2 months after streptozotocin [14]. By contrast, we did not found any protective effect of vitamin E on albuminuria of streptozotocin-induced diabetic rats [17]. Trachtman and coworkers [12] reported greater decline in renal function and accelerated glomerulosclerosis in diabetic rats treated with vitamin E. In the same study [12], dietary supplement with taurine 1% in drinking water for 1 year attenuated proteinuria and glomerulosclerosis in diabetic rats. Present study also demonstrates effective prevention of proteinuria by taurine at dose inhibiting lipid peroxidation. In addition, taurine totally abolished increases in glomerular TGF-b1 and fibronectin mRNA expression, supporting the previous long-term beneficial effect of taurine on diabetic glemerulosclerosis [12]. In spite of potent antioxidant effect of melatonin on brain, lung, and liver [22], no study has been conducted to investigate protective effect of melatonin on the kidney from oxidative damage. Melatonin at dose inhibiting lipid peroxidation prevented increased glomerular TGF-b1 and fibronectin mRNA expresssion and proteinuria, suggesting that beneficial effect of melatonin may result from its antioxidative property. A recent study [42] demonstrating that chronic prooxidant state increased renal TGF-b1 and extracellular matrix protein mRNA expression in the rats supports our results. In addition, high glucose produced more hydrogen peroxide by murine mesangial cells [15] and hydrogen peroxide increased TGF-b1 and fibronectin synthesis in mesangial cells [43]. The significance of lowered TGF-b1 and fibronectin mRNAs in the glomeruli of diabetic rats treated with melatonin below control re-
Antioxidants and diabetic glomerulopathy
mains to be defined in future studies investigating the long-term effects of melatonin on control as well as diabetic kidneys. Neither melatonin nor taurine has any effect on plasma glucose. Lack of correlation between proteinuria and plasma glucose but strong correlation between proteinuria and lipid peroxidation further suggest that oxidative stress associated with hyperglycemia has a role on increased expression of glomerular TGF-b1 and fibronectin mRNAs and proteinuria of diabetic rats. Abolishment of glomerular TGF-b1 and fibronectin mRNA increments and proteinuria in diabetic rats with 50% reduction of lipid peroxidation by melatonin and taurine suggest that oxidative stress above threshold is a pathogenic factor of diabetic nephropathy. The ability of antioxidants to prevent increases in glomerular TGF-b1 and fibronectin mRNAs in diabetic kidney is consistent with previous in vitro studies which demonstrated effective inhibition of high glucose-induced TGF-b1 and extracellular matrix protein synthesis [9 –11,44]. Activation of PKC, one of the major biochemical pathway inducing diabetic nephropathy [45], is sensitively regulated through redox changes in sulfhydryl groups of cystein-rich regions in PKC [46,47]. Antioxidants including vitamin E and taurine suppressed increases in PKC and TGF-b synthesis by mesangial cells in response to high glucose [44], suggesting antioxidants reduce the level of cellular oxidants and protect reactive protein sulfhydryls from oxidation. Vitamin E, but not taurine, may have direct effects on interaction of the PKC system with diacyl glycerol (DAG) , since it directly increase DAG kinase leading to inhibition of PKC activation [14,44]. On the other hand, our preliminary study [43] demonstrated that inhibition of PKC effectively blocked phorbol ester-, high glucose-, and H2O2-induced TGF-b1 and fibronectin synthesis by mesangial cells [43] and suggested modulatory role of PKC on oxidative glomerular injury in diabetes. Thus, coordinate interaction between oxidative stress and PKC under hyperglycemia may lead to increase in glomerular TGF-b1 and fibronectin mRNAs. In conclusion, our data demonstrate beneficial effects of melatonin and taurine on early changes in diabetic kidney and suggest that diabetic nephropathy associated with hyperglycemia is largely mediated by oxidative stress. Acknowledgements — We thank Professor Hi Bahl Lee at Hyonam Kidney Laboratory, Soon Chun Hyang University, for his continued interest in our work and constructive criticism and Dr. Sung Il Kim for providing cDNA probes for TGF-b1 and GAPDH. This work was supported in part by a grant from Korea Science and Engineering foundation (KOSEF 94-0403-07-01-3). Portions of this work were presented at the 30th Annual Meeting of American Society of Nephrology (San Antonio, TX, USA, 1997) and appeared in abstract form.
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ABBREVIATIONS
CR— control rats DR— diabetic rats GAPDH— glyceraldehyde-3-phosphate dehydrogenase LPO—lipid peroxides M—melatonin PCR—polymerase chain reaction PKC—protein kinase C T—taurine TGF-b1—transforming growth factor-b1