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Susceptibility of ageing kidney to quinidine
145
SUSCEPTIBILITY
OF AGEING
A. K. AGARWAL
KIDNEY
TO QUINIDINE
and S. S. RAO
Tavicolngy Research and Training Center, Depurtment ofsciences, John Jay College of‘CUNY, 445 W 59th Street, New York, NY 10019, USA
SUMMARY Male Fischer-344 rats aged 34 months and 30-32 months were used in this study. Quinidine in vitro reduced accumulation of organic ions, p-ammonium hippurate (PAH) and tetraethyl ammonium (TEA), inhibited oxygen consumption and increased LDH leakage, in renal cortical slices. High concentrations of quinidine in any of (2 and 3 mmol 1-l) produced overt toxicity and no age related differences the parameters measured were observed. But at lower concentrations significant age-related differences in kidney susceptibility to quinidine were evident. Administration of 75 mg kg-’ day-’ quinidine for 4 days caused exacerbated renal damage in senescent rats compared to young adults as demonstrated by greater elevation of blood urea nitrogen and serum creatinine, and greater inhibition of TEA uptake in renal cortical slices. These results establish significant age related differences in renal damage due to quinidine. KEYWOKDS: quinidine, kidney, ageing.
INTRODUCTION Quinidine is a weak basic drug obtained from cinchona bark and has been clinically used as an antiarrhythmic agent. Several cases of liver damage due to quinidine have been reported [l-3]. In patients with congestive heart failure, quinidine administration produced a decrease in glomerular filtration rate with a decrease in creatinine clearance and elevated levels of blood urea nitrogen (BUN) in some patients [4]. Altered pharmacokinetics and pharmacodynamics with ageing can cause a significant difference in toxic responses at the same dose level between young adults and old. Age-related variations in renal blood flow and glomerular filtration rate cause less efficient clearance of drugs. Enhanced susceptibility to drug-induced nephrotoxicity has been observed in ageing laboratory rats [S-7]. The augmented nephrotoxicity in ageing rats due to drugs and chemicals has been suggested to be due to age-related differences in metabolism, disposition, transport and excretion. The objective of this study was 1043-6618/94/020145~10/$08.00/0
0 1994 The Italian Pharmacological
Society
Pharmuwlogrcal
146
to examine the age-related responses of rat kidney.
effect
MATERIALS
of quinidine
Research,
on biochemical
Vol. 29, No. 2. 1994
and functional
AND METHODS
Male Fischer-344 rats aged 3-4 months were purchased from Charles River Breeding Laboratories (Wilmington, MA, USA). Rats aged 30-32 months were obtained from Smith Kline Beecham Research Labs, King of Prussia, PA, USA. These animals were also initially purchased from Charles River Breeding Laboratories. All animals were maintained in central animal facilities at ambient temperature and humidity under a 12 h dark and light cycle. The animals received commercial food and regular water ad lihitum. The animals were not monitored for food and water intake in the in vivo studies, it was assumed not to have affected the results observed. In vitro oxygen consumption in renal cortical slices The animals were killed by cervical dislocation. Kidneys were removed, decapsulated and cortical slices were prepared freehand. Renal cortical slices prepared freehand have previously been used as an in vitro model to study the toxic effect of various nephrotoxic chemicals [8,9]. The slices (So-100 mg) were incubated in Kreb’s Ringer bicarbonate buffer with lactic acid (pH 7.4) containing O-3 mM quinidine in a Dubnoff metabolic shaking water bath at 37°C for 90 min under constant supply of 95:5 oxygen/carbon dioxide. Following incubation, oxygen consumption in the slices was recorded polarographically using a Clarke’s oxygen electrode fitted to a Gilson’s oxygraph. Slices were removed, blotted and homogenized in a 5 ml of 0.1 M phosphate buffer (pH 7.4). Measurement of renal cortical slice oxygen consumption is an established method for determining slice viability and the effect of nephrotoxicants [ 10, 111. Lactic dehydrogenase in the incubation medium and in the slice homogenate was measured spectrophotometrically as increase in absorbance of NAD at 366 nm using pyruvate as the substrate. Percent leakage of the enzyme from slices into the incubation medium was calculated and used as an index of cytotoxic damage. In \litro measurement of the leakage of the cytosolic enzyme LDH from renal cortical slices has been previously used as an indicator of cytotoxic damage [ 11, 121. Slice accumulation ofp-aminohippurate (PAH) and tetraethylammonium (TEA) Renal cortical slices (50-100 mg) were incubated in a phosphate buffered medium (pH 7.4) containing O-3 mmol 1-l quinidine, 10 mmol 1-l lactate, 75 pmol 1-l [jH]PAH (0.1 pCi3H m I-‘) (5 Ci mmol-‘, New England Nuclear, Boston, MA, USA) and 20 pmol 1-l [‘“Cl TEA (0.1 @i ‘“C ml-‘) (4.5 mCi mmol-‘, New England Nuclear, Boston, MA, USA) at 37°C under 100% oxygen for 90 min in a Dubnoff metabolic shaking water bath [13]. Slices were removed following incubation, blotted, weighed, and homogenized in 3 ml of 10% TCA and the final volume brought up to 10 ml with distilled water. A 2 ml aliquot of the incubation medium was added to 3 ml of 10% TCA and the final volume made up to 10 ml. Samples were centrifuged, and the supernatants were assayed for [‘I-l] PAH and
Pharmacological
Research,
Vol. 29, No 2. 1994
[‘“Cl TEA activity in a liquid TEA in the slices was expressed
scintillation counter. Accumulation as slice to medium ratio, S/M.
147
of PAH
and
In vivo experiments Quinidine hydrochloride in normal saline was administered intraperitoneally at a dose of 75 mg kg’ day-’ for 4 days. Control animals received normal saline alone in the same volume (1 ml kg-‘). The animals were killed on day 5, kidneys were removed and decapsulated. Cortical slices were prepared freehand. Oxygen consumption in the slices from the left kidneys was measured immediately as described above. Accumulation of PAH/TEA in cortical slices from the right kidneys was determined as described above. Blood was collected from lower aorta for biochemical measurements. BUN, and serum creatinine were determined according to Sigma Technical Bulletins 535, and 555 respectively. Statistical analysis All data are expressed as meanfso. Student’s t-test and the analysis of variance followed by Duncan’s multiple range means test were used for individual and group comparisons. Values were considered to be significantly different at Ps 0.05.
RESULTS Effect of quinidine in vitro Renal cortical slice accumulation of organic anion PAH was significantly lower in naive old rats aged 30-32 months than in young adults of 3-4 months (Fig. 1). Addition of quinidine to the incubation media significantly depressed PAH accumulation at concentrations of 2 and 3 mmol 1-l in young adults and at 1 mmol 1-l and above in senescent rats. Renal cortical slices from senescent rats showed a greater degree of inhibition of PAH accumulation than the slices for young rats at concentrations of 1 and 2 mmol 1-l of quinidine. At 3 mmol 1-l overt toxicity was observed and no age related difference was evident. Renal cortical slice accumulation of TEA was lower in naive senescent rats than in young adults. Quinidine decreased TEA accumulation in both age groups in a concentration dependent manner at levels of 0.5 mmol 1-l and above (Fig. 2). The decrease in TEA accumulation due to quinidine was greater in senescent rats than in young rats at concentrations of 0.5 and 1 mmol 1-l quinidine but at higher concentrations no significant age related difference was noted. Figure 3 shows the effect of quinidine on renal cortical slice oxygen consumption. Oxygen consumption in the slices from naive senescent rat kidneys was significantly lower than the young rat kidneys. Quinidine significantly reduced oxygen consumption at 2 and 3 mmol 1-l in both age groups and no age related difference in response to quinidine was observed. At 1 mmol 1-l quinidine, renal cortical slices from young rats failed to show any effect on oxygen consumption but slices from senescent rats showed a 25% decrease although it was not statistically significant. Leakage of the cytosolic enzyme, LDH, increased at 2.0 and 3.0 mmol I-’
148
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0
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0
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3.0
Fig. 1. Effect of quinidine on renal cortical slice accumulation of PAH. Renal cortical slices from naive 3-4 (0) and 30-32 (m) months-old Fischer-344 rats were incubated for 90 min in a medium containing O-3 mmol 1-l quinidine. *Significant difference from zero concentration controls. tsignificant age-related difference from 3-4 month-old rats. Values are mcankso of four to five samples from different animals.
*
i-* t*
l_i 0.5 Quinidine
(mmol l_ ’1
Fig. 2. Effect of qu,inidine on renal cortical slice accumulation of TEA. 0 34 month-old rats; ?? 30-32 months. *Significant difference from respective zero concentration controls. tsignificant age-related differences from 34 month-old rats. Values are mea&x) of four to five samples.
Pharmacological
Research,
Vol.
29, No.
2, 1994
149
t LL
I
t
*
t
*t
I
I
1
Quinidine
(mm0l1~~)
Fig. 3. Effect of quinidine on renal cortical slice oxygen consumption. 0 3-4 month-old rats; ??30-32 months. Oxygen consumption was measured at various concentrations of quinidine. *Significant difference from respective zero concentration controls. TSignificant difference from 34 month-old rat slices. Values are meankso of four to five samples.
90
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60-
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(mm01
2. 0
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I_ ’1
Fig. 4. Effect of quinidine on LDH leakage from renal cortical slices. Cl 34 month-old rats; ?? 30-32 months. LDH was determined in the incubation medium and in the slices. Results are expressed as percent LDH leakage from the slices. *Significant difference from respective zero concentration controls. Values are meanfso of four to five samples.
Phnrmcrcolo~~wl
150
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Vol. 29. No. 2. 1994
15
12
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0 I-
Control
Lontrol
‘l’reated PAH Quinidine
1 reaRa
TEA ’for 4 days
75 mg kg-’ day
Fig. 5. Three-to-four-month-old (0) and 30-32 month-old (m) rats were treated with quinidine i.p. 75 mg kg-’ day-’ for 4 days and killed on day 5. In Gtuo accumulation of PAH and TEA was measured as described. Values are mean+so of four to five animals. *Significant difference at PiO.05 from respective age controls. tsignificant age-related difference from 34 monthold rats.
-L T
-I-
h-ebated
Control Quinidine Fig. 6. Effect
t L
*
75 mg kg-’ day-
’for 4 days
of quinidine on renal cortical slice oxygen consumption. 0 34 month-old rats; ??30-32 months. Values are mean&so of four to five animals. *Significant difference at PZ 0.05 from respective age controls. iSignificant age-related difference compared to 3-4 monthold rats.
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151
Control 3.500
Treated
(b)
2.800 3
I z 3 2.100-
: ‘a ‘Z 1.400 g & 0.700 -
*
0.000 Control Quinidine
75 mg kg
Treated
’day- ’for 4 days
Fig. 7. Effect of quinidine administration on (a) blood urea nitrogen and (b) serum creatinine. 0 3-4 month-old rats; W 30-32 months. Values are meat&o of four to five samples. *Significant difference at P10.05 from respective age controls. -/Significant age-related difference compared to 3-4 month-old rats.
quinidine in both age groups (Fig. 4) and no age related difference in response to quinidine was observed. At 1 mmol l-‘, no increase in LDH leakage was observed in renal cortical slices from young rats but a 35% increase in LDH leakage was noticed in slices from senescent rats. The increase was not statistically significant. Effect
of quinidine
in vivo
Control senescent rats showed significantly lower renal cortical slice accumulation of PAH and TEA than control young adult rats (Fig. 5). Intraperitoneal administration of quinidine at 75 mg kg-’ day-’ for 4 days resulted in significant inhibition of TEA accumulation in both age groups, with greater
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Phormacologic~al
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Vol. 29, No. 2, I994
inhibition in slices from old rats as compared to the young adults (Fig. 5). Accumulation of PAH was not altered by quinidine. Measurement of oxygen consumption indicated significant inhibition in renal cortical slices prepared from young rats but no significant change in slices from senescent rats after quinidine administration (Fig. 6). Oxygen consumption in cortical slices from control senescent rat kidneys was lower than that from control young rat kidneys. BUN levels increased significantly in senescent rats but not in the young, whereas serum creatinine levels were elevated in both age groups. Increases in serum creatinine levels were greater in senescent rats (Fig. 7).
DISCUSSION The results of this study suggest that quinidine can cause renal damage in young adult rats and senescent rats as indicated by increased serum creatinine levels, increased BUN levels, decreased ability of renal cortical slices to accumulate organic anions and cations and decreased renal cortical slice oxygen consumption. No other study indicating quinidine nephrotoxicity has been reported. Since quinidine is an organic cation, the inhibition of TEA transport by quinidine is expected because of competition for a common transport system. Wabner and Chen [14] reported that renal transport of PAH was reduced in senescent Fischer-344 rats and that this effect occurs at the receptor level. Quinidine has been demonstrated to inhibit PAH transport in isolated perfused renal tubules [15]. Our in vitro studies suggest a significant age related difference in the effect of quinidine on PAH and TEA transport at lower concentrations, but, at higher concentrations overt toxicity was observed and no age related differences were found. In viva administration of quinidine at 75 mg kg-’ day-’ for 4 days resulted in significant inhibition of TEA transport in both age groups but the inhibition was significantly greater in senescent rats suggesting that renal cortical tubules of senescent rats are more susceptible to quinidine than those of young adults. Age related susceptibility in renal cortical slice accumulation of organic ions due to several nephrotoxicants has been previously reported [5-71. Decreased oxygen consumption in renal cortical slices due to quinidine is suggestive of mitochondrial damage. Proverbio et ul. [16] found that the active extrusion of Na’ undergoing Na/K exchange and the active extrusion of Na’ with Cl- and water were diminished in renal cortical slices from old rats as compared to the young. The oxygen consumption associated with each of the two active mechanisms of Na’ extrusion was also diminished in the old rats. Disturbances in bioenergetic functions have been believed to play an important role in age related diminution of a variety of physiological functions. Several studies have suggested that the number of mitochondria in various tissues decreases during senescence [17, 181. Mitochondrial respiration has been shown to decrease with age [19, 201. Quinidine has been reported to affect mitochondrial function in the heart [21]. Quinidine has also been reported to decrease state three oxygen consumption, respiratory control index, ADP/O ratio and the rate of phosphorylation in dog heart mitochondria [22]. Our preliminary studies suggested that quinidine
Phur-mucdo,qir~ul
Ruveurc~h. Vol. 29, No. 2. 1994
IS3
inhibited respiratory control index in isolated renal cortical mitochondria in male Fischer-344 and Sprague-Dawley rats. Oxidative phosphorylation was significantly inhibited in a concentration-dependent manner [23]. The results of this study indicate that quinidine decreases in vitro oxygen consumption and increases LDH leakage at 2 and 3 mmol 1-l in both age groups. It should be noted, however, that at 1 mmol 1-l renal cortical slices from old rats showed a 25% decrease in oxygen consumption and no change in slices from young rats suggesting a mild age related difference. LDH leakage also increased at I mmol 1-l by 35% in slices from senescent rats and no change was observed in slices from young adult rats again suggesting enhanced toxicity in senescent rats. The results suggest that at 2 and 3 mmol 1-l quinidine causes an overt toxicity and therefore no significant differences between the young and the senescent are observed at these concentrations. At lower concentrations age related susceptibility becomes more obvious. While reduced oxygen consumption after in viva administration of quinidine was observed in renal cortical slices from young rats, the oxygen consumption in slices from senescent rats was not affected. This discrepancy between in vitro and in vivo results might be due to one or more of several factors. Renal blood flow and glomerular filtration rates decline progressively with old age to almost onehalf to two-thirds of those measured in young adults [24, 251, with the result that only a fraction of the quinidine administered is actually delivered to the tubules, thus the proximal tubules of the two age groups are exposed to different concentrations of quinidine. The total number of active nephrons is reduced in old age and the remaining nephrons undergo functional hypertrophy [26]. While functional hypertrophy by itself does not damage the organ function, the energy requirement of the individual cells is greatly enhanced. It is also possible that due to adaptive hypertrophic and the compensatory responses of the remaining nephrons the inhibitory effect of quinidine on oxygen consumption does not become evident. The significant injury in senescent rats after quinidine administration is demonstrated in the form of elevated BUN and serum creatinine levels. In young adults BUN levels were not affected and serum creatinine levels were slightly elevated suggesting that renal injury was less than in senescent rats. It is therefore important to emphasize that the toxicity parameters chosen for analysis are crucial in determining overall age-related differences. In summary, age related susceptibility to quinidine induced renal damage is demonstrated at lower concentrations. In \,ivo administration of quinidine caused exacerbated renal damage in senescent rats compared to young adults as demonstrated by greater levels of BUN and serum creatinine, and greater reduction in TEA transport in renal cortical slices.
ACKNOWLEDGEMENTS These studies were supported in part by funds from Smith Kline Beecham Pharmaceuticals, and were presented in part at the 32nd annual meeting of the Society of Toxicology held in New Orleans, LA, USA in March 1993.
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REFERENCES I. Knobler H, Levij IS, Gavish D, Chajek-Shaul T. Quinidine induced hepatitis. Arch Int Med 1986; 146: 526-S. 2. Deisseroth A, Morganroth J, Winokur S. Quinidine-induced liver disease. Ann Int Med 1972; 77: 595-7. 3. Handler SD, Hirsch NR, Haas K, Davidson FZ. Quinidine hepatitis. Arch Inr Med 1975; 135: 871-2. 4. Bellet S, Roman LR, Boza A. Relation between serum quinidine levels and renal function. Am .I Cardiol 1971; 27: 368-7 1. 5. Kyle ME, Kocis JJ. The effect of age on salicylate induced nephrotoxicity in male rats. Toxic01 Appl Pharmacol 1985; 81: 337-47. 6. Goldstein RS, Pasino DA, Hook JB. Cephaloridine nephrotoxicity in aging male Fischer344 rats. Toxicology 1986; 83: 43-53. 7. Miura K, Goldstein RS, Morgan DG, Pasino DA, Hewitt WR, Hook JB. Age related differences in susceptibility to renal ischemia in rats. Toxirol Appl Pharmacol 1987; 87: 284-96. 8. Goldstein RS, Pasino DA, Hewitt WR, Hook JB. Biochemical mechanisms of cephaloridine nephrotoxicity: time and concentration dependence of peroxidative injury. Toxic01 Appl Pharmacol 1986; 83: 261-70. 9. Tarloff JB, Goldstein RS, Silver AC, Hewitt WR, Hook JB. Intrinsic susceptibility of the kidney to acetaminophen toxicity in middle-aged rats. Toxicol Lett 1990; 52: 101-10. IO. Rose RC, Bianchi J, Schuette SA. Effective use of renal cortical slices in transport and metabolic studies. Biochim Biophys Acta 1985; 821: 431-36. 11. Ruegg CE, Gruschenka HI, Wolfgang A, Gandolfi J, Brendel K. Preparation and utilization of positional renal slices for in vitro nephrotoxicity studies. In: McQueen CA, ed. In vitro toxicology: model systems and methods. New Jersey: The Telford Press, 1989; 197-230. 12. Phelps JS, Gandolfi AJ, Brendel K, Dorr RT. Cisplatin nephrotoxicity: in vitro studies with precision cut rabbit renal cortical slices. Toxic01 Appl Pharmacol 1987; 90: 501-12. 13. Cross RJ, Taggart VJ. Renal tubular transport: accumulation of p-aminohippurate by rabbit kidney slices. Am J Physiol 1950; 161: 181-90. 14. Wabner CL, Chen TS. Aging changes in renal handling of p-aminohippurate. Am J Physiol 1987; 252: R87 1-7. 15. Dantzler WH, Brokl OH. Verapamil and quinidine effects on PAH transport by isolated perfused renal tubules. Am J Physiol 1984; 246: F188-F200. 16. Proverbio F, Proverbio T, Marin R. Ion transport and oxygen consumption in kidney cortex slices from young and old rats. Gerontology 1985; 31: 166-73. 17. Stocco DM, Huson JC. Quantitation of mitochondrial DNA and protein in the liver of Fischer-344 rats during aging. J Gerontol 1978; 33: 802-9. 18. Burns EM, Kruckeberg TW, Comerford LE, Buschman I. Thinning of capillary walls and declining numbers of mitochondria in the cerebral cortex of the aging primate, Macaca nemesstrins. J Gerontol 1979; 34: 642-50. 19. Chiu YJD, Richardson A. Effect of age on the function of mitochondria isolated from brain and heart tissue. Exp Gerontol 1980; 15: 511-17. 20. Horton AA, Spencer JA. Decline in respiratory control ratio of rat liver mitochondria in old age. Mech Ageing Dev 1981; 17: 253-9. 21. Bachman E, Weber E, Zbinden G. Biochemical mechanisms of quinidine cardiotoxicity. J Cardiovasc Pharmucol 1986; 8: 826-3 1. 22. Alto LE, Dhalla NS. Effects of some antiarrhymic agents on dog heart mitochondrial oxidative phosphorylation. Eur J Pharmacol 1979; 59: 31 I-14. 23. Rao SS, Agarwal AK. Strain differences in the effect of quinidine on renal mitochondrial function. Toxicologist 1993; 13: 207. 24. Davies DF, Shock NW. Age changes in glomerular filtration rate, effective renal plasma flow, and tubular excretory capacity in adult males. J C/in invest 1950; 29: 496-507. 25. Kappel B, Olsen S. Cortical interstitial tissue and sclerosed glomeruli in the normal human kidney, related to age and sex. Virchows Arch 1980; A387: 27 1-7. 26. Hayslett JP. Functional adaptation to reduction in renal mass. Physiol Rev 1979; 59: 117-64.
SUSCEPTIBILITY
OF AGEING
A. K. AGARWAL
KIDNEY
TO QUINIDINE
and S. S. RAO
Tavicolngy Research and Training Center, Depurtment ofsciences, John Jay College of‘CUNY, 445 W 59th Street, New York, NY 10019, USA
SUMMARY Male Fischer-344 rats aged 34 months and 30-32 months were used in this study. Quinidine in vitro reduced accumulation of organic ions, p-ammonium hippurate (PAH) and tetraethyl ammonium (TEA), inhibited oxygen consumption and increased LDH leakage, in renal cortical slices. High concentrations of quinidine in any of (2 and 3 mmol 1-l) produced overt toxicity and no age related differences the parameters measured were observed. But at lower concentrations significant age-related differences in kidney susceptibility to quinidine were evident. Administration of 75 mg kg-’ day-’ quinidine for 4 days caused exacerbated renal damage in senescent rats compared to young adults as demonstrated by greater elevation of blood urea nitrogen and serum creatinine, and greater inhibition of TEA uptake in renal cortical slices. These results establish significant age related differences in renal damage due to quinidine. KEYWOKDS: quinidine, kidney, ageing.
INTRODUCTION Quinidine is a weak basic drug obtained from cinchona bark and has been clinically used as an antiarrhythmic agent. Several cases of liver damage due to quinidine have been reported [l-3]. In patients with congestive heart failure, quinidine administration produced a decrease in glomerular filtration rate with a decrease in creatinine clearance and elevated levels of blood urea nitrogen (BUN) in some patients [4]. Altered pharmacokinetics and pharmacodynamics with ageing can cause a significant difference in toxic responses at the same dose level between young adults and old. Age-related variations in renal blood flow and glomerular filtration rate cause less efficient clearance of drugs. Enhanced susceptibility to drug-induced nephrotoxicity has been observed in ageing laboratory rats [S-7]. The augmented nephrotoxicity in ageing rats due to drugs and chemicals has been suggested to be due to age-related differences in metabolism, disposition, transport and excretion. The objective of this study was 1043-6618/94/020145~10/$08.00/0
0 1994 The Italian Pharmacological
Society
Pharmuwlogrcal
146
to examine the age-related responses of rat kidney.
effect
MATERIALS
of quinidine
Research,
on biochemical
Vol. 29, No. 2. 1994
and functional
AND METHODS
Male Fischer-344 rats aged 3-4 months were purchased from Charles River Breeding Laboratories (Wilmington, MA, USA). Rats aged 30-32 months were obtained from Smith Kline Beecham Research Labs, King of Prussia, PA, USA. These animals were also initially purchased from Charles River Breeding Laboratories. All animals were maintained in central animal facilities at ambient temperature and humidity under a 12 h dark and light cycle. The animals received commercial food and regular water ad lihitum. The animals were not monitored for food and water intake in the in vivo studies, it was assumed not to have affected the results observed. In vitro oxygen consumption in renal cortical slices The animals were killed by cervical dislocation. Kidneys were removed, decapsulated and cortical slices were prepared freehand. Renal cortical slices prepared freehand have previously been used as an in vitro model to study the toxic effect of various nephrotoxic chemicals [8,9]. The slices (So-100 mg) were incubated in Kreb’s Ringer bicarbonate buffer with lactic acid (pH 7.4) containing O-3 mM quinidine in a Dubnoff metabolic shaking water bath at 37°C for 90 min under constant supply of 95:5 oxygen/carbon dioxide. Following incubation, oxygen consumption in the slices was recorded polarographically using a Clarke’s oxygen electrode fitted to a Gilson’s oxygraph. Slices were removed, blotted and homogenized in a 5 ml of 0.1 M phosphate buffer (pH 7.4). Measurement of renal cortical slice oxygen consumption is an established method for determining slice viability and the effect of nephrotoxicants [ 10, 111. Lactic dehydrogenase in the incubation medium and in the slice homogenate was measured spectrophotometrically as increase in absorbance of NAD at 366 nm using pyruvate as the substrate. Percent leakage of the enzyme from slices into the incubation medium was calculated and used as an index of cytotoxic damage. In \litro measurement of the leakage of the cytosolic enzyme LDH from renal cortical slices has been previously used as an indicator of cytotoxic damage [ 11, 121. Slice accumulation ofp-aminohippurate (PAH) and tetraethylammonium (TEA) Renal cortical slices (50-100 mg) were incubated in a phosphate buffered medium (pH 7.4) containing O-3 mmol 1-l quinidine, 10 mmol 1-l lactate, 75 pmol 1-l [jH]PAH (0.1 pCi3H m I-‘) (5 Ci mmol-‘, New England Nuclear, Boston, MA, USA) and 20 pmol 1-l [‘“Cl TEA (0.1 @i ‘“C ml-‘) (4.5 mCi mmol-‘, New England Nuclear, Boston, MA, USA) at 37°C under 100% oxygen for 90 min in a Dubnoff metabolic shaking water bath [13]. Slices were removed following incubation, blotted, weighed, and homogenized in 3 ml of 10% TCA and the final volume brought up to 10 ml with distilled water. A 2 ml aliquot of the incubation medium was added to 3 ml of 10% TCA and the final volume made up to 10 ml. Samples were centrifuged, and the supernatants were assayed for [‘I-l] PAH and
Pharmacological
Research,
Vol. 29, No 2. 1994
[‘“Cl TEA activity in a liquid TEA in the slices was expressed
scintillation counter. Accumulation as slice to medium ratio, S/M.
147
of PAH
and
In vivo experiments Quinidine hydrochloride in normal saline was administered intraperitoneally at a dose of 75 mg kg’ day-’ for 4 days. Control animals received normal saline alone in the same volume (1 ml kg-‘). The animals were killed on day 5, kidneys were removed and decapsulated. Cortical slices were prepared freehand. Oxygen consumption in the slices from the left kidneys was measured immediately as described above. Accumulation of PAH/TEA in cortical slices from the right kidneys was determined as described above. Blood was collected from lower aorta for biochemical measurements. BUN, and serum creatinine were determined according to Sigma Technical Bulletins 535, and 555 respectively. Statistical analysis All data are expressed as meanfso. Student’s t-test and the analysis of variance followed by Duncan’s multiple range means test were used for individual and group comparisons. Values were considered to be significantly different at Ps 0.05.
RESULTS Effect of quinidine in vitro Renal cortical slice accumulation of organic anion PAH was significantly lower in naive old rats aged 30-32 months than in young adults of 3-4 months (Fig. 1). Addition of quinidine to the incubation media significantly depressed PAH accumulation at concentrations of 2 and 3 mmol 1-l in young adults and at 1 mmol 1-l and above in senescent rats. Renal cortical slices from senescent rats showed a greater degree of inhibition of PAH accumulation than the slices for young rats at concentrations of 1 and 2 mmol 1-l of quinidine. At 3 mmol 1-l overt toxicity was observed and no age related difference was evident. Renal cortical slice accumulation of TEA was lower in naive senescent rats than in young adults. Quinidine decreased TEA accumulation in both age groups in a concentration dependent manner at levels of 0.5 mmol 1-l and above (Fig. 2). The decrease in TEA accumulation due to quinidine was greater in senescent rats than in young rats at concentrations of 0.5 and 1 mmol 1-l quinidine but at higher concentrations no significant age related difference was noted. Figure 3 shows the effect of quinidine on renal cortical slice oxygen consumption. Oxygen consumption in the slices from naive senescent rat kidneys was significantly lower than the young rat kidneys. Quinidine significantly reduced oxygen consumption at 2 and 3 mmol 1-l in both age groups and no age related difference in response to quinidine was observed. At 1 mmol 1-l quinidine, renal cortical slices from young rats failed to show any effect on oxygen consumption but slices from senescent rats showed a 25% decrease although it was not statistically significant. Leakage of the cytosolic enzyme, LDH, increased at 2.0 and 3.0 mmol I-’
148
12
9
tI-tt* IiI 1 i
F L& 2
3
i;*
0
n”r*
:
0
0.5
Quinidine
2.0 (mmol 1~ ’1
3.0
Fig. 1. Effect of quinidine on renal cortical slice accumulation of PAH. Renal cortical slices from naive 3-4 (0) and 30-32 (m) months-old Fischer-344 rats were incubated for 90 min in a medium containing O-3 mmol 1-l quinidine. *Significant difference from zero concentration controls. tsignificant age-related difference from 3-4 month-old rats. Values are mcankso of four to five samples from different animals.
*
i-* t*
l_i 0.5 Quinidine
(mmol l_ ’1
Fig. 2. Effect of qu,inidine on renal cortical slice accumulation of TEA. 0 34 month-old rats; ?? 30-32 months. *Significant difference from respective zero concentration controls. tsignificant age-related differences from 34 month-old rats. Values are mea&x) of four to five samples.
Pharmacological
Research,
Vol.
29, No.
2, 1994
149
t LL
I
t
*
t
*t
I
I
1
Quinidine
(mm0l1~~)
Fig. 3. Effect of quinidine on renal cortical slice oxygen consumption. 0 3-4 month-old rats; ??30-32 months. Oxygen consumption was measured at various concentrations of quinidine. *Significant difference from respective zero concentration controls. TSignificant difference from 34 month-old rat slices. Values are meankso of four to five samples.
90
r
*
75 -
* g
60-
4 -ym A
45-
8
30-
*.
15
0 l--hi_
1 *
i
ti 0
0.5
1.0 Quinidine
(mm01
2. 0
4
3. 0
I_ ’1
Fig. 4. Effect of quinidine on LDH leakage from renal cortical slices. Cl 34 month-old rats; ?? 30-32 months. LDH was determined in the incubation medium and in the slices. Results are expressed as percent LDH leakage from the slices. *Significant difference from respective zero concentration controls. Values are meanfso of four to five samples.
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12
0
9 I-
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3I-
0 I-
Control
Lontrol
‘l’reated PAH Quinidine
1 reaRa
TEA ’for 4 days
75 mg kg-’ day
Fig. 5. Three-to-four-month-old (0) and 30-32 month-old (m) rats were treated with quinidine i.p. 75 mg kg-’ day-’ for 4 days and killed on day 5. In Gtuo accumulation of PAH and TEA was measured as described. Values are mean+so of four to five animals. *Significant difference at PiO.05 from respective age controls. tsignificant age-related difference from 34 monthold rats.
-L T
-I-
h-ebated
Control Quinidine Fig. 6. Effect
t L
*
75 mg kg-’ day-
’for 4 days
of quinidine on renal cortical slice oxygen consumption. 0 34 month-old rats; ??30-32 months. Values are mean&so of four to five animals. *Significant difference at PZ 0.05 from respective age controls. iSignificant age-related difference compared to 3-4 monthold rats.
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Control 3.500
Treated
(b)
2.800 3
I z 3 2.100-
: ‘a ‘Z 1.400 g & 0.700 -
*
0.000 Control Quinidine
75 mg kg
Treated
’day- ’for 4 days
Fig. 7. Effect of quinidine administration on (a) blood urea nitrogen and (b) serum creatinine. 0 3-4 month-old rats; W 30-32 months. Values are meat&o of four to five samples. *Significant difference at P10.05 from respective age controls. -/Significant age-related difference compared to 3-4 month-old rats.
quinidine in both age groups (Fig. 4) and no age related difference in response to quinidine was observed. At 1 mmol l-‘, no increase in LDH leakage was observed in renal cortical slices from young rats but a 35% increase in LDH leakage was noticed in slices from senescent rats. The increase was not statistically significant. Effect
of quinidine
in vivo
Control senescent rats showed significantly lower renal cortical slice accumulation of PAH and TEA than control young adult rats (Fig. 5). Intraperitoneal administration of quinidine at 75 mg kg-’ day-’ for 4 days resulted in significant inhibition of TEA accumulation in both age groups, with greater
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inhibition in slices from old rats as compared to the young adults (Fig. 5). Accumulation of PAH was not altered by quinidine. Measurement of oxygen consumption indicated significant inhibition in renal cortical slices prepared from young rats but no significant change in slices from senescent rats after quinidine administration (Fig. 6). Oxygen consumption in cortical slices from control senescent rat kidneys was lower than that from control young rat kidneys. BUN levels increased significantly in senescent rats but not in the young, whereas serum creatinine levels were elevated in both age groups. Increases in serum creatinine levels were greater in senescent rats (Fig. 7).
DISCUSSION The results of this study suggest that quinidine can cause renal damage in young adult rats and senescent rats as indicated by increased serum creatinine levels, increased BUN levels, decreased ability of renal cortical slices to accumulate organic anions and cations and decreased renal cortical slice oxygen consumption. No other study indicating quinidine nephrotoxicity has been reported. Since quinidine is an organic cation, the inhibition of TEA transport by quinidine is expected because of competition for a common transport system. Wabner and Chen [14] reported that renal transport of PAH was reduced in senescent Fischer-344 rats and that this effect occurs at the receptor level. Quinidine has been demonstrated to inhibit PAH transport in isolated perfused renal tubules [15]. Our in vitro studies suggest a significant age related difference in the effect of quinidine on PAH and TEA transport at lower concentrations, but, at higher concentrations overt toxicity was observed and no age related differences were found. In viva administration of quinidine at 75 mg kg-’ day-’ for 4 days resulted in significant inhibition of TEA transport in both age groups but the inhibition was significantly greater in senescent rats suggesting that renal cortical tubules of senescent rats are more susceptible to quinidine than those of young adults. Age related susceptibility in renal cortical slice accumulation of organic ions due to several nephrotoxicants has been previously reported [5-71. Decreased oxygen consumption in renal cortical slices due to quinidine is suggestive of mitochondrial damage. Proverbio et ul. [16] found that the active extrusion of Na’ undergoing Na/K exchange and the active extrusion of Na’ with Cl- and water were diminished in renal cortical slices from old rats as compared to the young. The oxygen consumption associated with each of the two active mechanisms of Na’ extrusion was also diminished in the old rats. Disturbances in bioenergetic functions have been believed to play an important role in age related diminution of a variety of physiological functions. Several studies have suggested that the number of mitochondria in various tissues decreases during senescence [17, 181. Mitochondrial respiration has been shown to decrease with age [19, 201. Quinidine has been reported to affect mitochondrial function in the heart [21]. Quinidine has also been reported to decrease state three oxygen consumption, respiratory control index, ADP/O ratio and the rate of phosphorylation in dog heart mitochondria [22]. Our preliminary studies suggested that quinidine
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inhibited respiratory control index in isolated renal cortical mitochondria in male Fischer-344 and Sprague-Dawley rats. Oxidative phosphorylation was significantly inhibited in a concentration-dependent manner [23]. The results of this study indicate that quinidine decreases in vitro oxygen consumption and increases LDH leakage at 2 and 3 mmol 1-l in both age groups. It should be noted, however, that at 1 mmol 1-l renal cortical slices from old rats showed a 25% decrease in oxygen consumption and no change in slices from young rats suggesting a mild age related difference. LDH leakage also increased at I mmol 1-l by 35% in slices from senescent rats and no change was observed in slices from young adult rats again suggesting enhanced toxicity in senescent rats. The results suggest that at 2 and 3 mmol 1-l quinidine causes an overt toxicity and therefore no significant differences between the young and the senescent are observed at these concentrations. At lower concentrations age related susceptibility becomes more obvious. While reduced oxygen consumption after in viva administration of quinidine was observed in renal cortical slices from young rats, the oxygen consumption in slices from senescent rats was not affected. This discrepancy between in vitro and in vivo results might be due to one or more of several factors. Renal blood flow and glomerular filtration rates decline progressively with old age to almost onehalf to two-thirds of those measured in young adults [24, 251, with the result that only a fraction of the quinidine administered is actually delivered to the tubules, thus the proximal tubules of the two age groups are exposed to different concentrations of quinidine. The total number of active nephrons is reduced in old age and the remaining nephrons undergo functional hypertrophy [26]. While functional hypertrophy by itself does not damage the organ function, the energy requirement of the individual cells is greatly enhanced. It is also possible that due to adaptive hypertrophic and the compensatory responses of the remaining nephrons the inhibitory effect of quinidine on oxygen consumption does not become evident. The significant injury in senescent rats after quinidine administration is demonstrated in the form of elevated BUN and serum creatinine levels. In young adults BUN levels were not affected and serum creatinine levels were slightly elevated suggesting that renal injury was less than in senescent rats. It is therefore important to emphasize that the toxicity parameters chosen for analysis are crucial in determining overall age-related differences. In summary, age related susceptibility to quinidine induced renal damage is demonstrated at lower concentrations. In \,ivo administration of quinidine caused exacerbated renal damage in senescent rats compared to young adults as demonstrated by greater levels of BUN and serum creatinine, and greater reduction in TEA transport in renal cortical slices.
ACKNOWLEDGEMENTS These studies were supported in part by funds from Smith Kline Beecham Pharmaceuticals, and were presented in part at the 32nd annual meeting of the Society of Toxicology held in New Orleans, LA, USA in March 1993.
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REFERENCES I. Knobler H, Levij IS, Gavish D, Chajek-Shaul T. Quinidine induced hepatitis. Arch Int Med 1986; 146: 526-S. 2. Deisseroth A, Morganroth J, Winokur S. Quinidine-induced liver disease. Ann Int Med 1972; 77: 595-7. 3. Handler SD, Hirsch NR, Haas K, Davidson FZ. Quinidine hepatitis. Arch Inr Med 1975; 135: 871-2. 4. Bellet S, Roman LR, Boza A. Relation between serum quinidine levels and renal function. Am .I Cardiol 1971; 27: 368-7 1. 5. Kyle ME, Kocis JJ. The effect of age on salicylate induced nephrotoxicity in male rats. Toxic01 Appl Pharmacol 1985; 81: 337-47. 6. Goldstein RS, Pasino DA, Hook JB. Cephaloridine nephrotoxicity in aging male Fischer344 rats. Toxicology 1986; 83: 43-53. 7. Miura K, Goldstein RS, Morgan DG, Pasino DA, Hewitt WR, Hook JB. Age related differences in susceptibility to renal ischemia in rats. Toxirol Appl Pharmacol 1987; 87: 284-96. 8. Goldstein RS, Pasino DA, Hewitt WR, Hook JB. Biochemical mechanisms of cephaloridine nephrotoxicity: time and concentration dependence of peroxidative injury. Toxic01 Appl Pharmacol 1986; 83: 261-70. 9. Tarloff JB, Goldstein RS, Silver AC, Hewitt WR, Hook JB. Intrinsic susceptibility of the kidney to acetaminophen toxicity in middle-aged rats. Toxicol Lett 1990; 52: 101-10. IO. Rose RC, Bianchi J, Schuette SA. Effective use of renal cortical slices in transport and metabolic studies. Biochim Biophys Acta 1985; 821: 431-36. 11. Ruegg CE, Gruschenka HI, Wolfgang A, Gandolfi J, Brendel K. Preparation and utilization of positional renal slices for in vitro nephrotoxicity studies. In: McQueen CA, ed. In vitro toxicology: model systems and methods. New Jersey: The Telford Press, 1989; 197-230. 12. Phelps JS, Gandolfi AJ, Brendel K, Dorr RT. Cisplatin nephrotoxicity: in vitro studies with precision cut rabbit renal cortical slices. Toxic01 Appl Pharmacol 1987; 90: 501-12. 13. Cross RJ, Taggart VJ. Renal tubular transport: accumulation of p-aminohippurate by rabbit kidney slices. Am J Physiol 1950; 161: 181-90. 14. Wabner CL, Chen TS. Aging changes in renal handling of p-aminohippurate. Am J Physiol 1987; 252: R87 1-7. 15. Dantzler WH, Brokl OH. Verapamil and quinidine effects on PAH transport by isolated perfused renal tubules. Am J Physiol 1984; 246: F188-F200. 16. Proverbio F, Proverbio T, Marin R. Ion transport and oxygen consumption in kidney cortex slices from young and old rats. Gerontology 1985; 31: 166-73. 17. Stocco DM, Huson JC. Quantitation of mitochondrial DNA and protein in the liver of Fischer-344 rats during aging. J Gerontol 1978; 33: 802-9. 18. Burns EM, Kruckeberg TW, Comerford LE, Buschman I. Thinning of capillary walls and declining numbers of mitochondria in the cerebral cortex of the aging primate, Macaca nemesstrins. J Gerontol 1979; 34: 642-50. 19. Chiu YJD, Richardson A. Effect of age on the function of mitochondria isolated from brain and heart tissue. Exp Gerontol 1980; 15: 511-17. 20. Horton AA, Spencer JA. Decline in respiratory control ratio of rat liver mitochondria in old age. Mech Ageing Dev 1981; 17: 253-9. 21. Bachman E, Weber E, Zbinden G. Biochemical mechanisms of quinidine cardiotoxicity. J Cardiovasc Pharmucol 1986; 8: 826-3 1. 22. Alto LE, Dhalla NS. Effects of some antiarrhymic agents on dog heart mitochondrial oxidative phosphorylation. Eur J Pharmacol 1979; 59: 31 I-14. 23. Rao SS, Agarwal AK. Strain differences in the effect of quinidine on renal mitochondrial function. Toxicologist 1993; 13: 207. 24. Davies DF, Shock NW. Age changes in glomerular filtration rate, effective renal plasma flow, and tubular excretory capacity in adult males. J C/in invest 1950; 29: 496-507. 25. Kappel B, Olsen S. Cortical interstitial tissue and sclerosed glomeruli in the normal human kidney, related to age and sex. Virchows Arch 1980; A387: 27 1-7. 26. Hayslett JP. Functional adaptation to reduction in renal mass. Physiol Rev 1979; 59: 117-64.