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Glutathione status, lipid peroxidation and kidney function in streptozotocin diabetic rats
Exp Toxic Patho11994; 46: 143-147 Gustav Fischer Verlag Jena
Institute of Pharmacology and Toxicology'), Department of Internal Medicine 21, Faculty of Medicine, Friedrich Schiller University Jena, Germany
Glutathione status, lipid peroxidation and kidney function in streptozotocin diabetic rats H. BRAUNLICH'), F. MARX2 ) and G. STEIN2) With 6 figures Received: July 3, 1993; Accepted: July 27, 1993
Address for correspondence: Prof. Dr. H. BRAuNLICH, Institut flir Pharmakologie und Toxikologie der Friedrich-SchillerUniversitat Jena, LobderstraBe 1, D-07743 Jena, Germany.
Key words: Streptozotocin nephropathy; Glutathione status; Lipid peroxidation; Electrolyte excretion; p-Aminohippurate transport; Proteinuria; Diabetes, rat; Kidney function, diabetic rats.
Summary In adult female rats diabetic nephropathy was induced by i.v. administration of streptozotocin (6 mg/lOO g b.w.). The animals survive for 3 weeks when very low daily doses of insulin (0.3 IV/animal) are administered. High blood urea concentrations and distinct proteinuria indicate the impairment of kidney function in streptozotocin diabetic rats. Streptozotocin induces mild polyuria and increased renal excretion of potassium; there is also an increase in renal excretion of administered p-aminohippurate. Three weeks after administration of streptozotocin the formation of lipid peroxides is increased in the kidney. At this time glutathione content (GSH, GSSG) is unchanged in liver and kidney of streptozotocin diabetic rats. Impairment of kidney function in streptozotocin diabetic rats can be prevented by daily supplementation with sufficient doses of insulin (about 3 IV/animal).
Introduction In a wide range of doses treatment with streptozotocin causes selective impairment of pancreatic islet cells (DEAN and MATTHEWS 1972). In rats intravenous injection of 2.5 to 7.5 mg streptozotocinllOO g b.w. is followed by destruction of islet [3-cells. Caused by deprivation of insulin hyperglycemia and glucosuria were expressed 2 or 3 days after administration of a single dose of streptozotocin (JUNOD et al. 1969). There are no direct toxic effects of streptozotocin on the kidney (ARISON et al. 1967). The effects of disturbed insulin production in streptozotocin treated rats shows similarities with diabetic nephropathy (MAURER et al. 1976). The fundamental lesion in streptozotocin initiated diabetic nephropathy is characterized by the glomerular ac-
cumulation of proteins as the consequence of the increased synthesis and the attenuated degradation of glomerular basement membrane proteins (MAURER et al. 1976; BROWNLEE and SPIRO 1979; COHEN and KLEIN 1979). Furthermore, the reduced degradation of glycosylated circulating plasma proteins is implicated in pathogenesis of glomerular protein accumulation in diabetic nephropathy (BROWNLEE et al. 1983). In this study consequences of streptozotocin induced diabetic nephropathy on various renal functions were characterized in adult rats. Influences on renal excretion of electrolytes and p-aminohippurate were measured. The glutathione content in liver and kidney was determined in diabetic rats; formation of lipid peroxides was measured in the same organs since no data are available regarding influences of streptozotocin administration on these parameters.
Material and methods Animals Experiments were performed in llO-day-old female WISTAR rats (Uje:WIST). The animals were housed under controlled conventional conditions (standard diet and tap water ad libitum; natural lighting; room temperature 22-25 DC; humidity> 50 %).
Experimental protocol The experimental design was developed by TESCHNER et al. 1989: Diabetic nephrophathy was provoked by the intravenous administration of a single dose of streptozotocin (6 mg/lOO g b.w.; Sigma Chemie GmbH, Deisenhofen, Germany). Exp Toxic Patho146 (1994) 2 143
Control rats, matched for age and weight at the time of streptozotocin administration, have received an equal volume of saline. The diabetic rats were separated into two groups. In the first group, good control of blood glucose level was intended being called "well controlled group". Blood glucose levels were monitored in whole blood samples obtained by retroorbital sinus puncture. The individually adjusted dose of insulin was about 3 IU/animal . day, s.c. (Ultralente insulin; Novo Industries, Copenhagen, Denmark). A second group consisted of "poorly controlled" diabetic rats, receiving 0.3 IU insulin/animal . day, s.c., to insure a small weight gain and high blood glucose levels. Characterization of kidney functions and of biochemical parameters in liver and kidney were performed three weeks after the induction of diabetes.
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Results In comparison with controls weight gain of 110-dayold female rats was not significantly modified in streptozotocin diabetic rats. There is a tendency to a higher body weight in well controlled and to a lower body weight in 144
Exp Toxic Patho146 (1994) 2
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Arithmetic means ±SEM were given. Statistically significant differences between controls and both groups of streptozotocin diabetic animals were proved, using Student's t-test (p:::; 0.01).
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Blood glucose: Concentration of glucose was measured using a modified glucoseoxidase method (Fermognost blood glucose test, Arzneimittelwerk Dresden). Blood urea nitrogen (BUN): Urea concentrations in plasma were determined spectrophotometric ally (CERIOTTI and SPANDRIO 1963). Proteins: Total protein content in urine samples was measured with the Coomassie-blue dye-binding reaction according to BRADFORD 1976. Sodium and potassium: These electrolytes were determined in urine samples from saline loaded rats (5 ml 0.9 % NaCl/100 g b.w., i.p.). Concentrations of sodium and potassium were detected by flame photometry. p-Aminohippurate (PAH): Measurements were performed in urine samples from rats loaded with PAH at the beginning of urine collection (250 mg/100 g b.w., i.p.). PAH was detected with the colorimetric method, introduced by BRATTON and MARSHALL 1939. Glutathione (GSH, GSSG): Determinations were performed in liver and kidney tissue. Reduced glutathione (GSH) was measured according to KRETZSCHMAR et al. 1989; oxidized glutathione (GSSG) was detected by the fluorimetric method introduced by HISSIN and HILF 1976. Lipid peroxides: As described by YAGI (1987) measurement of the content of thiobarbituric acid reactive substances in tissue samples from liver and kidney was used as an indicator of lipid peroxide concentration.
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Fig. 1. Weight gain and blood glucose level during 18 days following administration of streptozotocin (6 mg/100 g b.w.). Well controlled rats (3 IU insulin/animal· day) and poorly controlled diabetic rats (0.3 IU insulin/animal· day) were compared with untreated controls (all changes are statistically significant; p ~ 0.01); arithmetic means ± SEM.; n=20. .c
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Fig. 2. Blood urea nitrogen level and renal excretion of proteins in streptozotocin diabetic rats (black columns); comparison with well controlled rats (3 IU insulin/animal· day) and untreated controls; measurements were performed on day 21 after administration of a single dose of streptozotocin (6 mg/100 g b.w.); arithmetic means ± SEM.; asterisks indicate statistically significant differences to untreated controls (p ~ 0.01). poorly controlled diabetic rats compared with controls (fig. 1). As shown in the lower part of the same figure, relatively constant blood glucose levels can be measured both in controls and in well controlled diabetic rats. Following daily administration of insulin (3 IU/animal) the blood glucose level in streptozotocin diabetic rats is sta-
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Fig. 3. Urine volume and renal excretion of sodium and potassium in well controlled and poorly controlled streptozotocin diabetic rats (3 or 0.3 IV insulin/animal· day); comparison with untreated controls; see legend to fig. 2.
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Fig. 4. Influence of streptozotocin treatment (6 mg/lOO g b.w.) on renal excretion of PAH in well controlled and poorly controlled diabetic rats (3 or 0.3 IV insulin/animal· day, respectively); the urine volume excreted from PAH-Ioaded rats is also given; measurements were performed 21 days following administration of a single dose of streptozotocin; statistically significant diferences to untreated controls (open colums) were marked by asterisks (p ~ 0.01); n=6.
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Fig. 5. Content of GSH in liver and kidney from streptozotocin diabetic rats with well and poor control of blood glucose level (3 or 0.3 IV insulin/animal· day); measurements were performed 21 days after administration of a single dose of streptozotocin (6 mg/lOO g b.w.); arithmetic means ± S.E.M.; n = 6.
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tistically significantly lower than in controls. High levels of blood glucose can be measured in diabetic rats receiving very low daily doses of insulin (0.3 IV/animal). In poorly controlled streptozotocin diabetic rats the increase in blood urea nitrogen content and in renal excretion of proteins is distinct and statistically significant (fig. 2). In streptozotocin diabetic rats moderate polyuria connected with increased renal excretion of potassium can be measured. Treatment of these diabetic rats with high daily doses of insulin (3 IU/animal) reduces renal excretion of water, sodium and potassium in comparison with controls (fig. 3). In PAH-Ioaded rats there is an increase in renal excretion of PAH in poorly controlled animals and a decrease in well controlled streptozotocin diabetic rats (fig. 4). As also shown in this figure, there are similar changes in urine volumes in diabetic rats.
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Fig. 6. Concentration of lipid peroxides in liver and kidney of controls and streptozotocin diabetic rats; well controlled and poorly controlled animals were compared (3 or 0.3 IV insulin/animal· day, respectively); asterisks indicate statistically significant differences between controls and streptozotocin diabetic rats ( p ~ 0.01); measurements were performed 21 days following administration of streptozotocin; n = 6. Exp Toxic Patho146 (1994) 2
145
Remarkably, expression of diabetic nephropathy following administration of streptozotocin is not connected with any changes in GSH content in liver and kidney (fig. 5). The same is true regarding the oxidized glutathione (GSSG); these results are not shown. In streptozotocin diabetic rats the poor control of diabetes is followed by increased formation of lipid peroxides in the kidney (fig. 6); this increase can be prevented by daily treatment with high doses of insulin (3 IV/animal). As also shown in figure 6, streptozotocin does not influence the formation of lipid peroxides in liver tissue.
ment of the kidney is not connected with deprivation of glutathione. It cannot be excluded, that an increased utilization of glutathione is compensated by a higher rate of synthesis. Following administration of various nephrotoxins or in renal ischemia a low content of GSH in kidney and in several other organs can be measured (TORRES et al. 1986; . SLUSSER et al. 1990). It seems to be of interest, that streptozotocin does not reduce the glutathione content, whereas a distinct increase in formation of lipid peroxides take place. In principle, increased lipid peroxidation is connected with a high rate of glutathione consumption (SLUSSER et al. 1990; BREZIS 1992; DARGEL 1992). Discussion In kidney tissue, formation of lipid peroxides is increased in streptozotocin diabetic rats. It cannot be diffeAs shown from our data, single injection of streptozo- rentiated from our data, whether oxidation of phospholitocin causes diabetic nephropathy with high blood glu- pids is increased in the glomeruli or in tubular nephron cose levels. Poorly controlled streptozotocin diabetic rats segments. An increased formation of lipid peroxides can with high levels of blood urea nitrogen survive for more be observed both after ischemic or toxic impairment of kidney function (COJOCEL 1989; SCHILLER et al. 1991; than 3 weeks. At this time distinct signs of diabetic nephropathy are WEINBERG 1991; WOLGASTet aI.1991). There are no data expressed. As mentioned from several authors proteinu- from the literature regarding influences of streptozotocin ria is the consequence of glomerular alterations, like ac- treatment on lipid peroxidation. In diabetic rats treated with individually adjusted doses cumulation and reduced degradation of proteins (MAUof insulin blood glucose levels are lower than in controls. RER et al. 1976; BROWNLEE et al. 1983). An additional increase in renal excretion of proteins of tubular origin Some changes in kidney function could be caused by hyperglycemia (diminished excretion of sodium, potassium (BERNARD and LAUWERYS 1991) cannot be excluded. It seems unlikely, that the induced injury ofthe glome- and p-arninohippurate). As shown from this study, streptozotocin induced diaruli in streptozotocin treated rats causes an increase in glomerular filtration and in the filtered fraction of elec- betic nephropathy is connected with distinct signs of tutrolytes and xenobiotics. Therefore, polyuria in strepto- bular impairment. There are no signs for direct nephrotozotocin diabetic rats could be caused by an increase in xic effects of streptozotocin: The measured renal effects renal excretion of glucose and by disturbed reabsorption of streptozotocin treatment were completely prevented of sodium in tubular nephron segments. Polyuria is a sen- by daily administration of high doses of retard-insulin. sitive and early marker of tubular dysfunction (FENT et aI. The increase in blood glucose level and consecutive metabolic disorders are obviously the prerequisite for ex1988). The remarkable lack of an increased renal excretion of pression of streptozotocin nephropathy. sodium in our streptozotocin diabetic rats can be explained by the increased sodium-potassium-exchange, indicated by a distinct and statistically significant in- References crease in renal excretion of potassium. Impairment of kidney function is also indicated by the ARISON RN, CIACCIO EL, GLITZER MS, et al.: Light an electron microscopy of lesions in rats rendered diabetic with diminished renal excretion of PAH or the reduced PAH streptozotocin. Diabetes 1967; 16: 51-62. transport in renal cortical slices (BERNDT 1989). Remar- BENNETI WM, PLAMP CE, PARKER RA, et al.: Alterations in kably, renal excretion of p-arninohippurate is increased in organic ion transport induced by gentamicin nephrotoxirats with streptozotocin diabetes. As mentioned above, it city in the rat. J Lab Clin Med 1980; 95: 32-39. seems to be unlikely that the glomerularly filtered frac- BERNARD A, LAUWERYS RR: Proteinuria - Changes and metion of PAH is increased. Also in rats treated with various chanisms in toxic nephropathies. Crit Rev Toxico11991; 21: 373-405. nephrotoxins an increase in renal excretion of PAH could BERNDT WO: Potential involvement of renal transport mebe measured; the interpretation was, that an overwhelchanisms in nephrotoxicity. Toxicol Letters 1989; 46: ming compensation of the impaired PAH transporter 72-82. takes place (BENNETT et aI. 1980; KRAUL et al. 1991). This phenomenon can be observed following administra- BRADFORD MM: A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the tion of low doses of nephrotoxins or at the beginning of principle of protein dye binding. Anal Biochem 1976; repeated exposition to nephrotoxic agents. 72: 248-254. There is no influence of diabetic nephropathy in strep- BRATION AC, MARSHALL EH: A new coupling component tozotocin treated rats on the glutathione status in kidney for sulfonamide determination. J BioI Chem 1939; 128: and liver. Obviously the primarily glomerular impair537-550. 146
Exp Toxic Pathol46 (1994) 2
BREZIS M: Forefronts in nephrology - summary of the newer aspects of renal cell injury. Kidney Int 1992; 42: 523-539. BROWNLEE M, SPIRO RG: Glomerular basement membrane metabolism in the diabetic rat. Diabetes 1979; 28: 121-125. BROWNLEE M, VLASSARA H, CERAMI A: Non-enzymatic glycosylation reduces the susceptibility of fibrin to degradation by plasmin. Diabetes 1983; 32: 680-684. CERIOTTI G, SPANDRIO L: A spectrophotometric method for determination of urea. Clin Chim Acta 1963; 8: 295-299. COHEN MP, KLEIN CD: Glomerulopathy in rats with streptozotocin diabetes. J Exp Med 1979; 149: 623-631. COJOCEL C, BEUTER W, MAYER D: Lipid peroxidation: A possible mechanism of trichloroethylene-induced nephrotoxicity. Toxicology 1989; 55: 131-141. DARGEL R: Lipid peroxidation - a common pathogenetic mechanism. Exp Toxicol Patho11992; 44: 169-181. DEA PM, MATTHEWS EK: The biophysical properties of pancreatic islet cells; effect of diabetogenic agents. Diabetologia 1972; 8: 173-178. DONALDSON J, HEMMING R, LABELLA F: Vanadium exposure enhances lipid peroxidation in the kidney of rats and mice. Canad J Physiol Pharmacol1985; 63: 196-199. FENT K, MEYER E, ZBINDEN G: Nephrotoxicity screening in rats, a validation study. Arch Toxicol 1988; 61: 349-358. HISSIN PJ, HILF R: A fluorimetric method for determination of oxidized and reduced glutathione in tissue. Anal Biochern 1976; 74: 214-226. JUNOD A, STAUFFACHER W, RENOLD AE: Diabetogenic action of streptozotocin: Relationship of dose to metabolic response. J Clin Invest 1969; 48: 2129. KRAUL H, JAHN F, BRAuNLICH H: Nephrotoxic effect of
diethylene glycol (DEG) in rats. Exp Pathol 1991; 42: 27-32. KRETZSCHMAR M, FRANKE H, ZIMMERMANN T, et al.: Glutathione synthesis and export in experimental liver cirrhosis induced by thioazetamide: Relations to ultrastructural changes. Exp Path 1989; 36: 113-122. MAURER SM, STEFFES MW, MICHAEL AF, et al.: Studies of diabetic nephropathy in animals and man. Diabetes 1976; 25(SuppI.2): 850-857. SCHILLER HJ, ANDREONI KA, BULKLEY GB: Free radical ablation for the prevention of post-ischemic renal failure following renal transplantation. Klin Wschr 1991; 69: 1083-1094. SLUSSER SO, GROTYOHANN LW, MARTIN LF: Glutathione catabolism by ischemic rat kidney. Amer J Physiol 1990; 258: 1547-1553. TESCHNER M, SCHAEFER RM, SVARNAS A, et al.: Decreased proteinase activity in isolated glomeruli of streptozotocin diabetic rats. Amer J Nephro11989; 9: 464-469. TORRES AM, RODRIGUEZ JV, OCHOA JE, et al.: Rat kidney function related to tissue glutathione levels. Biochem Pharmacol 1986;35: 3355-3359. VERMEULEN NPE, BALDEW GS: The role of lipid peroxidation in the nephrotoxicity of cisplatin. Biochem Pharmacol 1992; 44: 1193-1200. WALKER RJ, DUGGIN GG: Drug nephrotoxicity. Ann Rev Pharmacol Toxicol 1988; 28: 331-346. WEINBERG JM: The cell biology of ischemic renal injury. KidneyIntI991;39:476-500. WOLGAST M, BAYATI A, HELLBERG 0, et al.: Oxygen radicals in postischemic damages in the kidney. Klin Wschr 1991;69: 1077-1082. Y AGI K: Lipid peroxide in human diseases. Chern Phys Lipids 1987: 45: 337-351.
Exp Toxic Patho146 (1994) 2
147
Institute of Pharmacology and Toxicology'), Department of Internal Medicine 21, Faculty of Medicine, Friedrich Schiller University Jena, Germany
Glutathione status, lipid peroxidation and kidney function in streptozotocin diabetic rats H. BRAUNLICH'), F. MARX2 ) and G. STEIN2) With 6 figures Received: July 3, 1993; Accepted: July 27, 1993
Address for correspondence: Prof. Dr. H. BRAuNLICH, Institut flir Pharmakologie und Toxikologie der Friedrich-SchillerUniversitat Jena, LobderstraBe 1, D-07743 Jena, Germany.
Key words: Streptozotocin nephropathy; Glutathione status; Lipid peroxidation; Electrolyte excretion; p-Aminohippurate transport; Proteinuria; Diabetes, rat; Kidney function, diabetic rats.
Summary In adult female rats diabetic nephropathy was induced by i.v. administration of streptozotocin (6 mg/lOO g b.w.). The animals survive for 3 weeks when very low daily doses of insulin (0.3 IV/animal) are administered. High blood urea concentrations and distinct proteinuria indicate the impairment of kidney function in streptozotocin diabetic rats. Streptozotocin induces mild polyuria and increased renal excretion of potassium; there is also an increase in renal excretion of administered p-aminohippurate. Three weeks after administration of streptozotocin the formation of lipid peroxides is increased in the kidney. At this time glutathione content (GSH, GSSG) is unchanged in liver and kidney of streptozotocin diabetic rats. Impairment of kidney function in streptozotocin diabetic rats can be prevented by daily supplementation with sufficient doses of insulin (about 3 IV/animal).
Introduction In a wide range of doses treatment with streptozotocin causes selective impairment of pancreatic islet cells (DEAN and MATTHEWS 1972). In rats intravenous injection of 2.5 to 7.5 mg streptozotocinllOO g b.w. is followed by destruction of islet [3-cells. Caused by deprivation of insulin hyperglycemia and glucosuria were expressed 2 or 3 days after administration of a single dose of streptozotocin (JUNOD et al. 1969). There are no direct toxic effects of streptozotocin on the kidney (ARISON et al. 1967). The effects of disturbed insulin production in streptozotocin treated rats shows similarities with diabetic nephropathy (MAURER et al. 1976). The fundamental lesion in streptozotocin initiated diabetic nephropathy is characterized by the glomerular ac-
cumulation of proteins as the consequence of the increased synthesis and the attenuated degradation of glomerular basement membrane proteins (MAURER et al. 1976; BROWNLEE and SPIRO 1979; COHEN and KLEIN 1979). Furthermore, the reduced degradation of glycosylated circulating plasma proteins is implicated in pathogenesis of glomerular protein accumulation in diabetic nephropathy (BROWNLEE et al. 1983). In this study consequences of streptozotocin induced diabetic nephropathy on various renal functions were characterized in adult rats. Influences on renal excretion of electrolytes and p-aminohippurate were measured. The glutathione content in liver and kidney was determined in diabetic rats; formation of lipid peroxides was measured in the same organs since no data are available regarding influences of streptozotocin administration on these parameters.
Material and methods Animals Experiments were performed in llO-day-old female WISTAR rats (Uje:WIST). The animals were housed under controlled conventional conditions (standard diet and tap water ad libitum; natural lighting; room temperature 22-25 DC; humidity> 50 %).
Experimental protocol The experimental design was developed by TESCHNER et al. 1989: Diabetic nephrophathy was provoked by the intravenous administration of a single dose of streptozotocin (6 mg/lOO g b.w.; Sigma Chemie GmbH, Deisenhofen, Germany). Exp Toxic Patho146 (1994) 2 143
Control rats, matched for age and weight at the time of streptozotocin administration, have received an equal volume of saline. The diabetic rats were separated into two groups. In the first group, good control of blood glucose level was intended being called "well controlled group". Blood glucose levels were monitored in whole blood samples obtained by retroorbital sinus puncture. The individually adjusted dose of insulin was about 3 IU/animal . day, s.c. (Ultralente insulin; Novo Industries, Copenhagen, Denmark). A second group consisted of "poorly controlled" diabetic rats, receiving 0.3 IU insulin/animal . day, s.c., to insure a small weight gain and high blood glucose levels. Characterization of kidney functions and of biochemical parameters in liver and kidney were performed three weeks after the induction of diabetes.
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Results In comparison with controls weight gain of 110-dayold female rats was not significantly modified in streptozotocin diabetic rats. There is a tendency to a higher body weight in well controlled and to a lower body weight in 144
Exp Toxic Patho146 (1994) 2
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10
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0
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Analytical methods
Arithmetic means ±SEM were given. Statistically significant differences between controls and both groups of streptozotocin diabetic animals were proved, using Student's t-test (p:::; 0.01).
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Blood glucose: Concentration of glucose was measured using a modified glucoseoxidase method (Fermognost blood glucose test, Arzneimittelwerk Dresden). Blood urea nitrogen (BUN): Urea concentrations in plasma were determined spectrophotometric ally (CERIOTTI and SPANDRIO 1963). Proteins: Total protein content in urine samples was measured with the Coomassie-blue dye-binding reaction according to BRADFORD 1976. Sodium and potassium: These electrolytes were determined in urine samples from saline loaded rats (5 ml 0.9 % NaCl/100 g b.w., i.p.). Concentrations of sodium and potassium were detected by flame photometry. p-Aminohippurate (PAH): Measurements were performed in urine samples from rats loaded with PAH at the beginning of urine collection (250 mg/100 g b.w., i.p.). PAH was detected with the colorimetric method, introduced by BRATTON and MARSHALL 1939. Glutathione (GSH, GSSG): Determinations were performed in liver and kidney tissue. Reduced glutathione (GSH) was measured according to KRETZSCHMAR et al. 1989; oxidized glutathione (GSSG) was detected by the fluorimetric method introduced by HISSIN and HILF 1976. Lipid peroxides: As described by YAGI (1987) measurement of the content of thiobarbituric acid reactive substances in tissue samples from liver and kidney was used as an indicator of lipid peroxide concentration.
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9
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time after streptozotoci n (days)
Fig. 1. Weight gain and blood glucose level during 18 days following administration of streptozotocin (6 mg/100 g b.w.). Well controlled rats (3 IU insulin/animal· day) and poorly controlled diabetic rats (0.3 IU insulin/animal· day) were compared with untreated controls (all changes are statistically significant; p ~ 0.01); arithmetic means ± SEM.; n=20. .c
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Fig. 2. Blood urea nitrogen level and renal excretion of proteins in streptozotocin diabetic rats (black columns); comparison with well controlled rats (3 IU insulin/animal· day) and untreated controls; measurements were performed on day 21 after administration of a single dose of streptozotocin (6 mg/100 g b.w.); arithmetic means ± SEM.; asterisks indicate statistically significant differences to untreated controls (p ~ 0.01). poorly controlled diabetic rats compared with controls (fig. 1). As shown in the lower part of the same figure, relatively constant blood glucose levels can be measured both in controls and in well controlled diabetic rats. Following daily administration of insulin (3 IU/animal) the blood glucose level in streptozotocin diabetic rats is sta-
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Fig. 3. Urine volume and renal excretion of sodium and potassium in well controlled and poorly controlled streptozotocin diabetic rats (3 or 0.3 IV insulin/animal· day); comparison with untreated controls; see legend to fig. 2.
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0
o 0.3 3.0 insulin(JU/animal)
Fig. 4. Influence of streptozotocin treatment (6 mg/lOO g b.w.) on renal excretion of PAH in well controlled and poorly controlled diabetic rats (3 or 0.3 IV insulin/animal· day, respectively); the urine volume excreted from PAH-Ioaded rats is also given; measurements were performed 21 days following administration of a single dose of streptozotocin; statistically significant diferences to untreated controls (open colums) were marked by asterisks (p ~ 0.01); n=6.
o
0.3 3.0
0 0.3 3.0 insulin (JU lani mal)
Fig. 5. Content of GSH in liver and kidney from streptozotocin diabetic rats with well and poor control of blood glucose level (3 or 0.3 IV insulin/animal· day); measurements were performed 21 days after administration of a single dose of streptozotocin (6 mg/lOO g b.w.); arithmetic means ± S.E.M.; n = 6.
01
~
200
liver
2
tistically significantly lower than in controls. High levels of blood glucose can be measured in diabetic rats receiving very low daily doses of insulin (0.3 IV/animal). In poorly controlled streptozotocin diabetic rats the increase in blood urea nitrogen content and in renal excretion of proteins is distinct and statistically significant (fig. 2). In streptozotocin diabetic rats moderate polyuria connected with increased renal excretion of potassium can be measured. Treatment of these diabetic rats with high daily doses of insulin (3 IU/animal) reduces renal excretion of water, sodium and potassium in comparison with controls (fig. 3). In PAH-Ioaded rats there is an increase in renal excretion of PAH in poorly controlled animals and a decrease in well controlled streptozotocin diabetic rats (fig. 4). As also shown in this figure, there are similar changes in urine volumes in diabetic rats.
"0
E
5III C1J 1J
100
x
0
L..
C1J
a. 1J
a.
.-
-
0 o
0.3
3.0
0 0.3 3.0 insulin (JU/animal)
Fig. 6. Concentration of lipid peroxides in liver and kidney of controls and streptozotocin diabetic rats; well controlled and poorly controlled animals were compared (3 or 0.3 IV insulin/animal· day, respectively); asterisks indicate statistically significant differences between controls and streptozotocin diabetic rats ( p ~ 0.01); measurements were performed 21 days following administration of streptozotocin; n = 6. Exp Toxic Patho146 (1994) 2
145
Remarkably, expression of diabetic nephropathy following administration of streptozotocin is not connected with any changes in GSH content in liver and kidney (fig. 5). The same is true regarding the oxidized glutathione (GSSG); these results are not shown. In streptozotocin diabetic rats the poor control of diabetes is followed by increased formation of lipid peroxides in the kidney (fig. 6); this increase can be prevented by daily treatment with high doses of insulin (3 IV/animal). As also shown in figure 6, streptozotocin does not influence the formation of lipid peroxides in liver tissue.
ment of the kidney is not connected with deprivation of glutathione. It cannot be excluded, that an increased utilization of glutathione is compensated by a higher rate of synthesis. Following administration of various nephrotoxins or in renal ischemia a low content of GSH in kidney and in several other organs can be measured (TORRES et al. 1986; . SLUSSER et al. 1990). It seems to be of interest, that streptozotocin does not reduce the glutathione content, whereas a distinct increase in formation of lipid peroxides take place. In principle, increased lipid peroxidation is connected with a high rate of glutathione consumption (SLUSSER et al. 1990; BREZIS 1992; DARGEL 1992). Discussion In kidney tissue, formation of lipid peroxides is increased in streptozotocin diabetic rats. It cannot be diffeAs shown from our data, single injection of streptozo- rentiated from our data, whether oxidation of phospholitocin causes diabetic nephropathy with high blood glu- pids is increased in the glomeruli or in tubular nephron cose levels. Poorly controlled streptozotocin diabetic rats segments. An increased formation of lipid peroxides can with high levels of blood urea nitrogen survive for more be observed both after ischemic or toxic impairment of kidney function (COJOCEL 1989; SCHILLER et al. 1991; than 3 weeks. At this time distinct signs of diabetic nephropathy are WEINBERG 1991; WOLGASTet aI.1991). There are no data expressed. As mentioned from several authors proteinu- from the literature regarding influences of streptozotocin ria is the consequence of glomerular alterations, like ac- treatment on lipid peroxidation. In diabetic rats treated with individually adjusted doses cumulation and reduced degradation of proteins (MAUof insulin blood glucose levels are lower than in controls. RER et al. 1976; BROWNLEE et al. 1983). An additional increase in renal excretion of proteins of tubular origin Some changes in kidney function could be caused by hyperglycemia (diminished excretion of sodium, potassium (BERNARD and LAUWERYS 1991) cannot be excluded. It seems unlikely, that the induced injury ofthe glome- and p-arninohippurate). As shown from this study, streptozotocin induced diaruli in streptozotocin treated rats causes an increase in glomerular filtration and in the filtered fraction of elec- betic nephropathy is connected with distinct signs of tutrolytes and xenobiotics. Therefore, polyuria in strepto- bular impairment. There are no signs for direct nephrotozotocin diabetic rats could be caused by an increase in xic effects of streptozotocin: The measured renal effects renal excretion of glucose and by disturbed reabsorption of streptozotocin treatment were completely prevented of sodium in tubular nephron segments. Polyuria is a sen- by daily administration of high doses of retard-insulin. sitive and early marker of tubular dysfunction (FENT et aI. The increase in blood glucose level and consecutive metabolic disorders are obviously the prerequisite for ex1988). The remarkable lack of an increased renal excretion of pression of streptozotocin nephropathy. sodium in our streptozotocin diabetic rats can be explained by the increased sodium-potassium-exchange, indicated by a distinct and statistically significant in- References crease in renal excretion of potassium. Impairment of kidney function is also indicated by the ARISON RN, CIACCIO EL, GLITZER MS, et al.: Light an electron microscopy of lesions in rats rendered diabetic with diminished renal excretion of PAH or the reduced PAH streptozotocin. Diabetes 1967; 16: 51-62. transport in renal cortical slices (BERNDT 1989). Remar- BENNETI WM, PLAMP CE, PARKER RA, et al.: Alterations in kably, renal excretion of p-arninohippurate is increased in organic ion transport induced by gentamicin nephrotoxirats with streptozotocin diabetes. As mentioned above, it city in the rat. J Lab Clin Med 1980; 95: 32-39. seems to be unlikely that the glomerularly filtered frac- BERNARD A, LAUWERYS RR: Proteinuria - Changes and metion of PAH is increased. Also in rats treated with various chanisms in toxic nephropathies. Crit Rev Toxico11991; 21: 373-405. nephrotoxins an increase in renal excretion of PAH could BERNDT WO: Potential involvement of renal transport mebe measured; the interpretation was, that an overwhelchanisms in nephrotoxicity. Toxicol Letters 1989; 46: ming compensation of the impaired PAH transporter 72-82. takes place (BENNETT et aI. 1980; KRAUL et al. 1991). This phenomenon can be observed following administra- BRADFORD MM: A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the tion of low doses of nephrotoxins or at the beginning of principle of protein dye binding. Anal Biochem 1976; repeated exposition to nephrotoxic agents. 72: 248-254. There is no influence of diabetic nephropathy in strep- BRATION AC, MARSHALL EH: A new coupling component tozotocin treated rats on the glutathione status in kidney for sulfonamide determination. J BioI Chem 1939; 128: and liver. Obviously the primarily glomerular impair537-550. 146
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BREZIS M: Forefronts in nephrology - summary of the newer aspects of renal cell injury. Kidney Int 1992; 42: 523-539. BROWNLEE M, SPIRO RG: Glomerular basement membrane metabolism in the diabetic rat. Diabetes 1979; 28: 121-125. BROWNLEE M, VLASSARA H, CERAMI A: Non-enzymatic glycosylation reduces the susceptibility of fibrin to degradation by plasmin. Diabetes 1983; 32: 680-684. CERIOTTI G, SPANDRIO L: A spectrophotometric method for determination of urea. Clin Chim Acta 1963; 8: 295-299. COHEN MP, KLEIN CD: Glomerulopathy in rats with streptozotocin diabetes. J Exp Med 1979; 149: 623-631. COJOCEL C, BEUTER W, MAYER D: Lipid peroxidation: A possible mechanism of trichloroethylene-induced nephrotoxicity. Toxicology 1989; 55: 131-141. DARGEL R: Lipid peroxidation - a common pathogenetic mechanism. Exp Toxicol Patho11992; 44: 169-181. DEA PM, MATTHEWS EK: The biophysical properties of pancreatic islet cells; effect of diabetogenic agents. Diabetologia 1972; 8: 173-178. DONALDSON J, HEMMING R, LABELLA F: Vanadium exposure enhances lipid peroxidation in the kidney of rats and mice. Canad J Physiol Pharmacol1985; 63: 196-199. FENT K, MEYER E, ZBINDEN G: Nephrotoxicity screening in rats, a validation study. Arch Toxicol 1988; 61: 349-358. HISSIN PJ, HILF R: A fluorimetric method for determination of oxidized and reduced glutathione in tissue. Anal Biochern 1976; 74: 214-226. JUNOD A, STAUFFACHER W, RENOLD AE: Diabetogenic action of streptozotocin: Relationship of dose to metabolic response. J Clin Invest 1969; 48: 2129. KRAUL H, JAHN F, BRAuNLICH H: Nephrotoxic effect of
diethylene glycol (DEG) in rats. Exp Pathol 1991; 42: 27-32. KRETZSCHMAR M, FRANKE H, ZIMMERMANN T, et al.: Glutathione synthesis and export in experimental liver cirrhosis induced by thioazetamide: Relations to ultrastructural changes. Exp Path 1989; 36: 113-122. MAURER SM, STEFFES MW, MICHAEL AF, et al.: Studies of diabetic nephropathy in animals and man. Diabetes 1976; 25(SuppI.2): 850-857. SCHILLER HJ, ANDREONI KA, BULKLEY GB: Free radical ablation for the prevention of post-ischemic renal failure following renal transplantation. Klin Wschr 1991; 69: 1083-1094. SLUSSER SO, GROTYOHANN LW, MARTIN LF: Glutathione catabolism by ischemic rat kidney. Amer J Physiol 1990; 258: 1547-1553. TESCHNER M, SCHAEFER RM, SVARNAS A, et al.: Decreased proteinase activity in isolated glomeruli of streptozotocin diabetic rats. Amer J Nephro11989; 9: 464-469. TORRES AM, RODRIGUEZ JV, OCHOA JE, et al.: Rat kidney function related to tissue glutathione levels. Biochem Pharmacol 1986;35: 3355-3359. VERMEULEN NPE, BALDEW GS: The role of lipid peroxidation in the nephrotoxicity of cisplatin. Biochem Pharmacol 1992; 44: 1193-1200. WALKER RJ, DUGGIN GG: Drug nephrotoxicity. Ann Rev Pharmacol Toxicol 1988; 28: 331-346. WEINBERG JM: The cell biology of ischemic renal injury. KidneyIntI991;39:476-500. WOLGAST M, BAYATI A, HELLBERG 0, et al.: Oxygen radicals in postischemic damages in the kidney. Klin Wschr 1991;69: 1077-1082. Y AGI K: Lipid peroxide in human diseases. Chern Phys Lipids 1987: 45: 337-351.
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