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Amphotericin B decreases adenylyl cyclase activity and aquaporin-2 expression in rat kidney
Amphotericin B decreases adenylyl cyclase activity and aquaporin-2 expression in rat kidney SOO WAN KIM, CHUNG HO YEUM, SUNMI KIM, YOONWHA OH, KI CHUL CHOI, and JONGUN LEE GWANGJU, KOREA
The present study was intended to examine whether the amphotericin-induced urinary concentration defect can be related to an altered regulation of aquaporin (AQP) water channels in the kidney. Male Sprague-Dawley rats were injected with amphotericin B (6 mg/kg/d, IP) for 21 days. The protein expression of AQP1-3, Gsα, and adenylyl cyclase was determined in the kidney. To further specify the primary point of dysregulation of AQP channels that are activated by the arginine vasopressin/cyclic adenosine monophosphate (AVP/cAMP) pathway, different components of adenylyl cyclase complex were separately examined for their cAMP-generating activities. Amphotericin treatment resulted in kidney failure associated with decreased tubular water reabsorption and increased urinary flow rate. The expression of AQP2 proteins was significantly decreased in the outer medulla and inner medulla but not in the cortex. The expression of AQP2 proteins in the membrane fraction changed in parallel with that in the cytoplasmic fraction, suggesting a preserved targeting. Neither the expression of AQP1 nor that of AQP3 was significantly affected in the cortex, outer medulla, or inner medulla. The cAMP generation in response to AVP or sodium fluoride was decreased, whereas that to forskolin was not significantly altered. The expression of Gsα proteins was decreased in the inner medulla, whereas that of adenylyl cyclase VI remained unaltered. These findings indicate that the amphotericin-induced urinary concentration defect may in part be causally related to a reduced abundance of AQP2 channels in the kidney. It is also suggested that the primary impairment in the pathway leading to the activation of AQP channels that are regulated by the AVP/cAMP pathway lies at the level of G proteins. (J Lab Clin Med 2001;138:243-9)
Abbreviations: AQP = aquaporin; AVP = arginine vasopressin; cAMP = cyclic adenosine monophosphate; EDTA = ethylenediaminetetraacetic acid; HD = high density; LD = low density; PMSF = phenylmethylsulfonyl fluoride; TBST = Tris-based saline buffer containing 0.1% Tween-20
From the Departments of Physiology and Internal Medicine, Chonnam National University Medical School, and Chonnam National University Research Institute of Medical Sciences. Supported by research grants from Chonnam National University (2000) and the Hormone Research Center (2001G0301). Submitted for publication January 24, 2001; revision submitted May 29, 2001; accepted June 11, 2001. Reprint requests: JongUn Lee, MD, Department of Physiology, Chonnam National University Medical School, 5 Hak-Dong, Gwangju 501-746, Republic of Korea. Copyright © 2001 by Mosby, Inc. 0022-2143/2001 $35.00 + 0 5/1/117826 doi:10.1067/mlc.2001.117826
A
mphotericin B has been widely used for severe life-threatening fungal infections.1 However, its therapeutic usefulness may be limited by the potential nephrotoxicity, which is manifested by reduced glomerular filtration rate and tubular dysfunction.2,3 The most prominent features associated with amphotericin-induced nephropathy are polyuria and urinary concentration defect, which have been attributed in experimental and clinical studies to an inability of the collecting duct to respond adequately to AVP.4-6 However, detailed mechanisms underlying the urinary concentration defect caused by amphotericin have not been established. 243
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Fig 1. Expression of AQP2 water channels in the cortex (C), outer medulla (OM), and inner medulla (IM) of the kidney of control and amphotericin-treated rats. Representative immunoblots of AQP2 and densitometric data are shown. White columns indicate controls, shaded columns indicate amphotericin-treated. Each column depicts the mean ± SEM of 6 rats. *P < .05, **P < .01 as compared with controls.
Fig 2. Expression of AQP2 water channels in the membrane and cytoplasmic fractions of the kidney. Immunoblots of AQP2 in the HD and LD fractions are shown. White column indicates control, shaded column indicates amphotericin-treated. Each column represents the mean ± SEM of 8 rats.
The recent discovery of AQP water channels has advanced our understanding of water transport and AVP action in the kidney. They play an important role in the urinary concentration through generation of medullary hypertonicity and regulation of collecting duct water permeability.7 Among the multiple isoforms of the AQP family, AQP1 is highly expressed in the proximal tubule and descending thin limb.8 The critical role of AQP1 has been confirmed in transgenic mice lacking AQP1 that are unable to concentrate the urine and are severely dehydrated.9 The abundance of AQP2 is highly expressed in the principal cell of the collecting duct.10,11 It is regulated in the short-term and long-term by the AVP/cAMP pathway to increase osmotic water reabsorption. Short-term regulation of AQP2 channels occurs as a result of an exocytic insertion of the cytoplasmic AQP2 vesicles into the apical membrane,12 whereas the long-term effect is to increase the total abundance of AQP2 proteins.13 Water reabsorption across the basolateral membrane of the collecting duct is in turn mediated by AQP3 and AQP4.14,15
An altered role of AQP channels in the kidney has been observed in association with a decreased urinary concentration ability, such as acute ischemic kidney failure,16 urinary tract obstruction,17 gentamicininduced nephropathy,18 chronic kidney failure induced by surgical renal mass reduction,19 and cisplatininduced nephropathy.20 The present study was intended to examine the hypothesis that an altered regulation of AQP channels in the kidney accounts for the amphotericin-induced urinary concentration defect. The expression of AQP1-3 proteins and the catalytic activity of adenylyl cyclase in the kidney were determined in rats treated with amphotericin. METHODS Animals and urine collection. Male Sprague-Dawley rats (200 to 220 g) were used. They were kept in accordance with the Institutional Guidelines for Laboratory Animal Care and Use. Amphotericin B (Fungizone; Bristol-Myers Squibb, Princeton, NJ) was injected intraperitoneally (6 mg/kg/d for 21 days). The rats injected with vehicle during the same period served as controls. On the next day of the last treatment, the rats were anesthetized with ketamine (50 mg/kg, IP), and
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Fig 3. Expression of AQP1 water channels in the cortex (C), outer medulla (OM), and inner medulla (IM) of the kidney. Representative immunoblots of AQP1 and densitometric data are shown. White columns indicate controls, shaded columns indicate amphotericintreated. Each column shows the mean ± SEM of 6 rats.
Fig 4. Expression of AQP3 water channels in the cortex (C), outer medulla (OM), and inner medulla (IM) of the kidney. Representative immunoblots of AQP3 and densitometric data are shown. White columns indicate controls, shaded columns indicate amphotericintreated. Each column shows the mean ± SEM of 6 rats.
the urinary bladder was cannulated to collect urine samples. An elapsing period of 12 to 1 hour was allowed after the surgical preparation was made. The urine was then collected for 1 hour, and at the end of this collection arterial blood was taken to determine creatinine clearance and free water reabsorption, in which free water reabsorption (TcH2O) was calculated by using the following equation: TcH2O = V (Uosm/Posm – 1), where V stands for urine volume, Uosm stands for urine osmolality, and Posm stands for plasma osmolality. Protein preparation and Western blot analysis. After the rats were treated with amphotericin, the kidneys were removed under ketamine anesthesia. The kidneys were rapidly frozen and kept at –70°C until assayed. The cortex, outer medulla, and inner medulla from the frozen kidneys were dissected and then homogenized at 3000 rpm in a solution containing 250 mmol/L sucrose, 1 mmol/L EDTA, 0.1 mmol/L PMSF, and 10 mmol/L Tris-HCl buffer, pH 7.6. Large tissue debris and nuclear fragments were removed by two low-speed spins in succession (1000g, 10 minutes; 10,000g, 10 minutes). Protein samples were loaded and electrophoretically size-separated with a discontinuous system consisting of 12.5% polyacrylamide resolving gel and 5% polyacrylamide stacking gel. The proteins were then electrophoretically transferred to
a nitrocellulose membrane at 40 V for 3 hours. The membranes were washed in TBST (pH 7.4) containing 0.1% Tween-20 (Amresco, Solon, OH), blocked with 5% nonfat milk in TBST for 1 hour, and incubated with affinity-purified anti-rabbit polyclonal AQP1 (diluted 1:750), AQP2 (1:750), and AQP3 (1:200) antibodies (Alomone Lab; Jerusalem, Israel), heteromeric G-protein subunit Gsα (1:1000) (Calbiochem-Novabiochem; San Diego, CA), or type VI adenylyl cyclase (1:200) (Santa Cruz, Santa Cruz, CA) in 2% nonfat milk/TBST for 1 hour at room temperature. The membranes were then incubated with a horseradish peroxidase–labeled goat anti-rabbit immunoglobulin G (1:1200) in 2% nonfat milk in TBST for 1 hour. The bound antibody was detected by enhanced chemiluminescence (Amersham, Little Chalfont, Buckinghamshire, UK) on hyperfilm. Relative protein levels were determined by analyzing the signals of autoradiograms with the transmitter scanning videodensitometer (Bioneer, Cheongwon, Korea). Differential centrifugation. AQP2 targeting was assessed by comparing the magnitude of its expression in the membrane-enriched fraction and in the cytoplasmic fraction.21 The kidney homogenate was centrifuged at low-speed spins (1000g for 10 minutes) to remove cell debris and nuclear fragments. It was then centrifuged at 17,000g for 20 minutes to
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Fig 5. cAMP production provoked by AVP, sodium fluoride, and forskolin in the inner medulla. Open circles indicate controls, solid circles indicate amphotericin-treated. Each point represents the mean ± SEM of six experiments. *P < .05, **P < .01, ***P < .001, as compared with controls.
yield membrane-enriched pellets (HD fraction). The supernatant was centrifuged again at 100,000g for 1 hour to obtain a cytoplasmic pellet (LD fraction). An altered targeting of AQP2 channels was determined by the ratio of HD/LD, where a diminished ratio implies a decrease. Membrane preparation and adenylyl cyclase activity.
The inner medulla was dissected and homogenized in icecold buffer (50 mmol/L Tris-HCl, pH 8.0, containing 1 mmol/L EDTA, 0.2 mmol/L PMSF, and 250 mmol/L sucrose) and centrifuged at 1000g and 100,000g in succession. The resulting pellet was used as membrane preparation. Protein concentrations were measured by bicinchonic acid assay kit (BioRad, Hercules, CA). The adenylyl cyclase activity in the membrane preparation was determined as described previously.16 Adenylyl cyclase consists of three major parts: receptor, G protein, and catalytic unit.22 The G protein acts as a transducer and sends a signal from the hormone-occupied receptor to the catalytic unit.23 The catalytic unit then induces enzymatic activity responsible for adenosine triphosphate hydrolysis to yield cAMP. To examine these parts separately, AVP was used to activate the V2 receptor, sodium fluoride was used to stimulate adenylyl cyclase in a receptor-independent but G-protein–dependent manner, and forskolin was used to probe the catalytic unit of adenylyl cyclase complex. The reaction was started by adding the membrane fraction, of which protein content was 10 µg for the inner medulla, in 100 µL working solution (50 mmol/L Tris-HCl, pH 7.6, containing 1 mmol/L adenosine triphosphate, 20 mmol/L phosphocreatine, 0.2 mg/mL creatine phosphokinase, 6.4 mmol/L MgCl2, 1 mmol/L 3-isobutyl-1-methylxanthine, and 0.02 mmol/L guanosine triphosphate). The reaction was then stopped after 15 minutes by application of cold solution consisting of 50 mmol/L sodium acetate (pH 5.0) and was centrifuged at 1000g for 10 minutes at 4°C. cAMP in the supernatant was measured by equilibrated radioimmunoassay. Iodinated 2´-O-monosuccinyl-adenosine 3´,5´-cyclic monophosphate tyrosyl methyl ester (iodine
125–labeled cAMP) was prepared as described by previous investigators.24 Standards or samples were taken up in a final volume of 100 µL of 50 mmol/L sodium acetate buffer (pH 4.8). A 100 µL sample of dilute cAMP antiserum (Calbiochem-Novabiochem, San Diego, CA) and iodine 125–labeled cAMP (10,000 cpm/100 µL) were added and incubated at 4°C for 15 hours. The bound form was separated from the free form by charcoal suspension, and the supernatant was counted in a gamma counter (Packard, Meriden, CT). All samples in one experiment were analyzed in a single assay. Nonspecific binding was <2.0%. The 50% intercept point was at 16.5 ± 0.8 fmol/tube (n = 10). The intraand interassay coefficients of variation were 5.0% ± 1.2% (n = 10) and 9.6% ± 1.9% (n = 10), respectively. Results were expressed as moles of cAMP generated per milligram of protein per minute. Drugs and statistical analysis. Drugs were purchased from Sigma Chemical Co, St Louis, MO, unless stated otherwise. Results are expressed as mean ± SEM. The statistical significance of differences between the groups was determined with an unpaired t test. RESULTS Kidney functional parameters. Table I summarizes the kidney function data. After the treatment with amphotericin, plasma creatinine clearance was decreased and serum creatinine levels were increased. Urinary flow rates were significantly increased, while the urine-toplasma ratio of osmolality and tubular free water reabsorption were decreased. Expression of AQP water channels. The expression of AQP proteins was determined in the cortex, outer medulla, and inner medulla of the kidney. The antiAQP1 antibody recognized 29-kd and 35- to 50-kd bands corresponding to nonglycosylated and glycosylated AQP1, respectively. The anti-AQP2 antibody recognized 29-kd and 35- to 50-kd bands corre-
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Fig 6. Immunoblots of Gsα proteins in the inner medulla. Representative immunoblots and densitometric data are shown. White column indicates controls, shaded column indicates amphotericin-treated. Each column depicts the mean ± SEM of 6 rats. *P < .05 as compared with controls.
sponding to nonglycosylated and glycosylated AQP2, respectively. The anti-AQP3 antibody recognized 27kd and 33- to 40-kd bands corresponding to nonglycosylated and glycosylated AQP3, respectively. After the treatment with amphotericin, the expression of AQP2 was decreased in the outer medulla and inner medulla while not in the cortex (Fig 1). It was decreased in parallel in the membrane-enriched and cytoplasmic fractions (Fig 2). Neither the expression of AQP1 nor that of AQP3 was significantly altered in the cortex, outer medulla, or inner medulla (Figs 3 and 4). Adenylyl cyclase activity. The inner medulla was examined for its adenylyl cyclase activity (Fig 5). After the treatment with amphotericin, the cAMP generation in response to AVP was decreased in the inner medulla. The cAMP generation stimulated by sodium fluoride was also significantly blunted by amphotericin, whereas that provoked by forskolin was not significantly affected. Protein expression of Gsα and adenylyl cyclase VI. Fig 6 shows immunoblots of Gsα proteins expressed in the inner medulla. The anti-Gsα antibody recognized a doublet at 50 kd and at 45 kd. The treatment with amphotericin significantly decreased the expression of Gsα proteins. Fig 7 shows its immunoblots of type VI adenylyl cyclase. The antibody recognized a broad band around 160 kd that was not significantly affected by the amphotericin treatment.
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Fig 7. Immunoblots of type VI adenylyl cyclase in the inner medulla. White column indicates controls, shaded column indicates amphotericin-treated. Each column depicts the mean ± SEM of 6 rats.
Table I. Renal functional parameters in control and amphotericin-treated rats Control (n = 6)
Pcr (mg/dL) Ccr (mL/min) Urinary flow rate (µL/h) Posm (mOsm/kg H2O) Uosm (mOsm/kg H2O) (U/P)osm TcH2O (µL/min/kg)
0.41 1.34 215.6 289.8 1758.4 6.07 69.7
± ± ± ± ± ± ±
0.03 0.15 21.4 9.7 138.4 0.5 14.5
Amphotericin (n = 5)
0.96 0.51 758.7 321.2 627.9 1.95 37.4
± ± ± ± ± ± ±
0.07* 0.12† 103.2* 8.6‡ 56.3† 0.3† 11.7‡
Values are expressed as mean ± SEM. n, Number of rats; Pcr, plasma creatinine; Ccr, creatinine clearance; Posm, plasma osmolality; Uosm, urine osmolality; (U/P)osm, urine-to-plasma ratio of osmolality; TcH2O, solute-free water reabsorption. *P < .01 versus control. †P < .001 versus control. ‡P < .05 versus control.
DISCUSSION
Treatment with amphotericin resulted in a polyuric kidney failure. The serum creatinine level was increased along with a decrease in its renal clearance. Urine osmolality and tubular free water reabsorption were also decreased, indicating an impaired concentrating ability. These findings are in accord with those in patients treated with amphotericin, in which the impaired urinary concentration may represent nephrogenic diabetes insipidus with polyuria and hypotonic urine.5,6 It has also been shown that amphotericin decreases the stimulatory action of the AVP/cAMP
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pathway on water permeability in the inner medullary collecting duct in rats.25 However, the downstream of the AVP/cAMP pathway has not been explored in amphotericin-induced nephropathy. It has been known that the AVP/cAMP pathway has long-term and short-term regulatory effects on AQP2 water channels in the collecting duct.12,13 The present study showed that the total abundance of AQP2 proteins was decreased by amphotericin. The decrease was parallel in the membrane-enriched and cytoplasmic fractions, suggesting a preserved targeting. Similar findings were noted in rats with such acquired forms of nephrogenic diabetes insipidus syndromes as cisplatin-induced nephropathy,20 lithium-induced nephropathy,26 and chronic kidney failure induced by surgical renal mass reduction.19 Nevertheless, it is likely that there may have been an initial transient impairment of AQP2 targeting after amphotericin treatment, while a reduced total abundance may prevail in the long run. The cAMP generation in response to AVP was significantly blunted after treatment with amphotericin. Therefore the primary site of impairment was further determined by the generation of cAMP with different pharmacologic tools. The adenylyl cyclase activity in response to sodium fluoride, which activates the adenylyl cyclase in a receptor-independent but G-protein– dependent manner,27 was attenuated after amphotericin treatment. In contrast, the cAMP generation stimulated by forskolin, which directly activates the catalytic unit of adenylyl cyclase,28 was not affected. These findings indicate that the G protein may be the site primarily injured by amphotericin. The speculation is substantiated by the reduced expression of Gsα proteins, with no significant alterations of adenylyl cyclase VI expression. A primary derangement of G protein may in turn result in a failure to adequately stimulate the catalytic unit of the adenylyl cyclase and hence the generation of cAMP. AQP1 is extremely abundant in the proximal tubule and descending thin limb, and AQP1 gene knockout mice have demonstrated 80% to 90% reduction of osmotic water permeability therein.9,29 It appears to provide the chief route of water reabsorption in the proximal nephron. An important role of AQP3 in urinary concentration has also been suggested by a remarkable polyuria and polydipsia in AQP3-null mice.30 In contrast to the expression of AQP2, however, neither the expression of AQP1 nor that of AQP3 was significantly altered by amphotericin. This finding suggests that AQP1 and AQP3 channels are not involved in the amphotericin-induced impairment of urinary concentration. It also suggests that different regulatory mechanisms exist between AQP1/AQP3 and AQP2.
In summary, the expression of AQP2 water channels was decreased in the kidney after treatment with amphotericin in association with a polyuric kidney failure. The decreased AQP2 expression may at least in part be causally related to the impairment of urinary concentration. On the other hand, the primary impairment in the pathway leading to the activation of AQP channels that are activated by the AVP/cAMP system may lie at the level of G proteins.
REFERENCES
1. Gallis HA, Drew RH, Pickard WW. Amphotericin B: 30 years of clinical experience. Rev Infect Dis 1990;12:308-29. 2. Cheng JT, Witty RT, Robinson RR, Yarger WE. Amphotericin B nephrotoxicity: increased renal resistance and tubule permeability. Kidney Int 1982;22:626-33. 3. Burgess JL, Birchall R. Nephrotoxicity of amphotericin B, with emphasis on changes in tubular function. Am J Med 1972;53:77-84. 4. Gouge TH, Andriole VT. An experimental model of amphotericin B nephrotoxicity with renal tubular acidosis. J Lab Clin Med 1971;73:713-24. 5. Barbour GL, Straub KD, O’Neal BL, Leatherman JW. Vasopressin-resistance nephrogenic diabetes insipidus. A result of amphotericin B therapy. Arch Intern Med 1979;193: 86-8. 6. Douglas JB, Healy JK. Nephrotoxic effects of amphotericin B including renal tubular acidosis. Am J Med 1969;46:15462. 7. Nielsen S, Kwon TH, Christensen BM, Promeneur D, Frokiaer J, Marples D. Physiology and pathophysiology of renal aquaporins. J Am Soc Nephrol 1999;10:647-63. 8. Nielsen S, Smith BL, Christensen EI, Knepper MA, Agre P. CHIP28 water channels are localized in constitutively waterpermeable segments of the nephron. J Cell Biol 1993;120: 371-83. 9. Ma T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels. J Biol Chem 1998;273:4296-9. 10. Fushimi K, Uchida S, Hara Y, Hirata Y, Marumo F, Sasaki S. Cloning and expression of apical membrane water channel of rat kidney collecting tubule. Nature 1993;361: 549-52. 11. Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, Harris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci USA 1993;90:11663-7. 12. Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, Knepper MA. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc Natl Acad Sci USA 1995;92:1013-7. 13. Terris J, Ecelbarger CA, Nielsen S, Knepper MA. Long-term regulation of four renal aquaporins in rat. Am J Physiol 1996;271:F414-22. 14. Nielsen S, Agre P. The aquaporin family of water channels in kidney. Kidney Int 1995;48:1057-68. 15. Knepper MA. The aquaporin family of molecular water channels. Proc Natl Acad Sci USA 1994;91:6255-8. 16. Kim SW, Jeon YS, Lee J, Kang DG, Kook H, Ahn KY, et al.
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Diminished adenylate cyclase activity and aquaporin 2 expression in acute renal failure rats. Kidney Int 2000;57: 1643-50. Kim SW, Cho SH, Oh BS, Yeum CH, Choi KC, Ahn KY, et al. Diminished renal expression of aquaporin water channels in rats with experimental bilateral ureteral obstruction. J Am Soc Nephrol. In press. Lee JU, Yoo KS, Kang DG, Kim SW, Choi KC. Gentamicin decreases the abundance of aquaporin water channels in rat kidney. Jpn J Pharmacol 2001;85:391-8. Kwon TH, Frøkiaer J, Knepper MA, Nielsen S. Reduced AQP1, -2, and -3 levels in kidneys of rats with CRF induced by surgical reduction in renal mass. Am J Physiol 1998; 275:F724-41. Kim SW, Lee JU, Nah MY, Kang DG, Ahn KY, Lee HS, et al. Cisplatin decreases the abundance of aquaporin water channels in rat kidney. J Am Soc Nephrol 2001;12: 875-82. Marples D, Knepper MA, Christensen EI, Nielsen S. Redistribution of aquaporin-2 water channels induced by vasopressin in rat kidney inner medullary collecting duct. Am J Physiol 1995;269:C655-64. Gilman AG. Guanine nucleotide-binding regulatory proteins and dual control of adenylate cyclase. J Clin Invest 1984; 73:1-4. Rodbell M. The role of hormone receptors and GTP-
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regulatory proteins in membrane transduction. Nature 1980; 284:17-22. Steiner AL, Kipnis DM, Utiger R, Parker C. Radioimmunoassay for the measurement of adenosine 3´,5´-cyclic phosphate. Proc Natl Acad Sci USA 1969;64:367-73. Yano Y, Monteiro JL, Seguro AC. Effect of amphotericin B on water and urea transport in the inner medullary collecting duct. J Am Soc Nephrol 1994;5:68-74. Marples D, Christensen S, Christensen EI, Ottosen PD, Nielsen S. Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla. J Clin Invest 1995;95:1838-45. Sternweis PC, Gilman AG. Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride. Proc Natl Acad Sci USA 1982;79:4888-91. Seamon KB, Daly JW. Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein. J Biol Chem 1981;256:9799-801. Schnermann J, Chou CL, Ma T, Traynor T, Knepper MA, Verkman AS. Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice. Proc Natl Acad Sci USA 1998;95:9660-4. Ma T, Song Y, Yang B, Gillespie A, Carlson EJ, Epstein CJ, et al. Nephrogenic diabetes insipidus in mice lacking aquaporin-3 water channels. Proc Natl Acad Sci USA 2000;97: 4386-91.
The present study was intended to examine whether the amphotericin-induced urinary concentration defect can be related to an altered regulation of aquaporin (AQP) water channels in the kidney. Male Sprague-Dawley rats were injected with amphotericin B (6 mg/kg/d, IP) for 21 days. The protein expression of AQP1-3, Gsα, and adenylyl cyclase was determined in the kidney. To further specify the primary point of dysregulation of AQP channels that are activated by the arginine vasopressin/cyclic adenosine monophosphate (AVP/cAMP) pathway, different components of adenylyl cyclase complex were separately examined for their cAMP-generating activities. Amphotericin treatment resulted in kidney failure associated with decreased tubular water reabsorption and increased urinary flow rate. The expression of AQP2 proteins was significantly decreased in the outer medulla and inner medulla but not in the cortex. The expression of AQP2 proteins in the membrane fraction changed in parallel with that in the cytoplasmic fraction, suggesting a preserved targeting. Neither the expression of AQP1 nor that of AQP3 was significantly affected in the cortex, outer medulla, or inner medulla. The cAMP generation in response to AVP or sodium fluoride was decreased, whereas that to forskolin was not significantly altered. The expression of Gsα proteins was decreased in the inner medulla, whereas that of adenylyl cyclase VI remained unaltered. These findings indicate that the amphotericin-induced urinary concentration defect may in part be causally related to a reduced abundance of AQP2 channels in the kidney. It is also suggested that the primary impairment in the pathway leading to the activation of AQP channels that are regulated by the AVP/cAMP pathway lies at the level of G proteins. (J Lab Clin Med 2001;138:243-9)
Abbreviations: AQP = aquaporin; AVP = arginine vasopressin; cAMP = cyclic adenosine monophosphate; EDTA = ethylenediaminetetraacetic acid; HD = high density; LD = low density; PMSF = phenylmethylsulfonyl fluoride; TBST = Tris-based saline buffer containing 0.1% Tween-20
From the Departments of Physiology and Internal Medicine, Chonnam National University Medical School, and Chonnam National University Research Institute of Medical Sciences. Supported by research grants from Chonnam National University (2000) and the Hormone Research Center (2001G0301). Submitted for publication January 24, 2001; revision submitted May 29, 2001; accepted June 11, 2001. Reprint requests: JongUn Lee, MD, Department of Physiology, Chonnam National University Medical School, 5 Hak-Dong, Gwangju 501-746, Republic of Korea. Copyright © 2001 by Mosby, Inc. 0022-2143/2001 $35.00 + 0 5/1/117826 doi:10.1067/mlc.2001.117826
A
mphotericin B has been widely used for severe life-threatening fungal infections.1 However, its therapeutic usefulness may be limited by the potential nephrotoxicity, which is manifested by reduced glomerular filtration rate and tubular dysfunction.2,3 The most prominent features associated with amphotericin-induced nephropathy are polyuria and urinary concentration defect, which have been attributed in experimental and clinical studies to an inability of the collecting duct to respond adequately to AVP.4-6 However, detailed mechanisms underlying the urinary concentration defect caused by amphotericin have not been established. 243
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Fig 1. Expression of AQP2 water channels in the cortex (C), outer medulla (OM), and inner medulla (IM) of the kidney of control and amphotericin-treated rats. Representative immunoblots of AQP2 and densitometric data are shown. White columns indicate controls, shaded columns indicate amphotericin-treated. Each column depicts the mean ± SEM of 6 rats. *P < .05, **P < .01 as compared with controls.
Fig 2. Expression of AQP2 water channels in the membrane and cytoplasmic fractions of the kidney. Immunoblots of AQP2 in the HD and LD fractions are shown. White column indicates control, shaded column indicates amphotericin-treated. Each column represents the mean ± SEM of 8 rats.
The recent discovery of AQP water channels has advanced our understanding of water transport and AVP action in the kidney. They play an important role in the urinary concentration through generation of medullary hypertonicity and regulation of collecting duct water permeability.7 Among the multiple isoforms of the AQP family, AQP1 is highly expressed in the proximal tubule and descending thin limb.8 The critical role of AQP1 has been confirmed in transgenic mice lacking AQP1 that are unable to concentrate the urine and are severely dehydrated.9 The abundance of AQP2 is highly expressed in the principal cell of the collecting duct.10,11 It is regulated in the short-term and long-term by the AVP/cAMP pathway to increase osmotic water reabsorption. Short-term regulation of AQP2 channels occurs as a result of an exocytic insertion of the cytoplasmic AQP2 vesicles into the apical membrane,12 whereas the long-term effect is to increase the total abundance of AQP2 proteins.13 Water reabsorption across the basolateral membrane of the collecting duct is in turn mediated by AQP3 and AQP4.14,15
An altered role of AQP channels in the kidney has been observed in association with a decreased urinary concentration ability, such as acute ischemic kidney failure,16 urinary tract obstruction,17 gentamicininduced nephropathy,18 chronic kidney failure induced by surgical renal mass reduction,19 and cisplatininduced nephropathy.20 The present study was intended to examine the hypothesis that an altered regulation of AQP channels in the kidney accounts for the amphotericin-induced urinary concentration defect. The expression of AQP1-3 proteins and the catalytic activity of adenylyl cyclase in the kidney were determined in rats treated with amphotericin. METHODS Animals and urine collection. Male Sprague-Dawley rats (200 to 220 g) were used. They were kept in accordance with the Institutional Guidelines for Laboratory Animal Care and Use. Amphotericin B (Fungizone; Bristol-Myers Squibb, Princeton, NJ) was injected intraperitoneally (6 mg/kg/d for 21 days). The rats injected with vehicle during the same period served as controls. On the next day of the last treatment, the rats were anesthetized with ketamine (50 mg/kg, IP), and
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Fig 3. Expression of AQP1 water channels in the cortex (C), outer medulla (OM), and inner medulla (IM) of the kidney. Representative immunoblots of AQP1 and densitometric data are shown. White columns indicate controls, shaded columns indicate amphotericintreated. Each column shows the mean ± SEM of 6 rats.
Fig 4. Expression of AQP3 water channels in the cortex (C), outer medulla (OM), and inner medulla (IM) of the kidney. Representative immunoblots of AQP3 and densitometric data are shown. White columns indicate controls, shaded columns indicate amphotericintreated. Each column shows the mean ± SEM of 6 rats.
the urinary bladder was cannulated to collect urine samples. An elapsing period of 12 to 1 hour was allowed after the surgical preparation was made. The urine was then collected for 1 hour, and at the end of this collection arterial blood was taken to determine creatinine clearance and free water reabsorption, in which free water reabsorption (TcH2O) was calculated by using the following equation: TcH2O = V (Uosm/Posm – 1), where V stands for urine volume, Uosm stands for urine osmolality, and Posm stands for plasma osmolality. Protein preparation and Western blot analysis. After the rats were treated with amphotericin, the kidneys were removed under ketamine anesthesia. The kidneys were rapidly frozen and kept at –70°C until assayed. The cortex, outer medulla, and inner medulla from the frozen kidneys were dissected and then homogenized at 3000 rpm in a solution containing 250 mmol/L sucrose, 1 mmol/L EDTA, 0.1 mmol/L PMSF, and 10 mmol/L Tris-HCl buffer, pH 7.6. Large tissue debris and nuclear fragments were removed by two low-speed spins in succession (1000g, 10 minutes; 10,000g, 10 minutes). Protein samples were loaded and electrophoretically size-separated with a discontinuous system consisting of 12.5% polyacrylamide resolving gel and 5% polyacrylamide stacking gel. The proteins were then electrophoretically transferred to
a nitrocellulose membrane at 40 V for 3 hours. The membranes were washed in TBST (pH 7.4) containing 0.1% Tween-20 (Amresco, Solon, OH), blocked with 5% nonfat milk in TBST for 1 hour, and incubated with affinity-purified anti-rabbit polyclonal AQP1 (diluted 1:750), AQP2 (1:750), and AQP3 (1:200) antibodies (Alomone Lab; Jerusalem, Israel), heteromeric G-protein subunit Gsα (1:1000) (Calbiochem-Novabiochem; San Diego, CA), or type VI adenylyl cyclase (1:200) (Santa Cruz, Santa Cruz, CA) in 2% nonfat milk/TBST for 1 hour at room temperature. The membranes were then incubated with a horseradish peroxidase–labeled goat anti-rabbit immunoglobulin G (1:1200) in 2% nonfat milk in TBST for 1 hour. The bound antibody was detected by enhanced chemiluminescence (Amersham, Little Chalfont, Buckinghamshire, UK) on hyperfilm. Relative protein levels were determined by analyzing the signals of autoradiograms with the transmitter scanning videodensitometer (Bioneer, Cheongwon, Korea). Differential centrifugation. AQP2 targeting was assessed by comparing the magnitude of its expression in the membrane-enriched fraction and in the cytoplasmic fraction.21 The kidney homogenate was centrifuged at low-speed spins (1000g for 10 minutes) to remove cell debris and nuclear fragments. It was then centrifuged at 17,000g for 20 minutes to
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Fig 5. cAMP production provoked by AVP, sodium fluoride, and forskolin in the inner medulla. Open circles indicate controls, solid circles indicate amphotericin-treated. Each point represents the mean ± SEM of six experiments. *P < .05, **P < .01, ***P < .001, as compared with controls.
yield membrane-enriched pellets (HD fraction). The supernatant was centrifuged again at 100,000g for 1 hour to obtain a cytoplasmic pellet (LD fraction). An altered targeting of AQP2 channels was determined by the ratio of HD/LD, where a diminished ratio implies a decrease. Membrane preparation and adenylyl cyclase activity.
The inner medulla was dissected and homogenized in icecold buffer (50 mmol/L Tris-HCl, pH 8.0, containing 1 mmol/L EDTA, 0.2 mmol/L PMSF, and 250 mmol/L sucrose) and centrifuged at 1000g and 100,000g in succession. The resulting pellet was used as membrane preparation. Protein concentrations were measured by bicinchonic acid assay kit (BioRad, Hercules, CA). The adenylyl cyclase activity in the membrane preparation was determined as described previously.16 Adenylyl cyclase consists of three major parts: receptor, G protein, and catalytic unit.22 The G protein acts as a transducer and sends a signal from the hormone-occupied receptor to the catalytic unit.23 The catalytic unit then induces enzymatic activity responsible for adenosine triphosphate hydrolysis to yield cAMP. To examine these parts separately, AVP was used to activate the V2 receptor, sodium fluoride was used to stimulate adenylyl cyclase in a receptor-independent but G-protein–dependent manner, and forskolin was used to probe the catalytic unit of adenylyl cyclase complex. The reaction was started by adding the membrane fraction, of which protein content was 10 µg for the inner medulla, in 100 µL working solution (50 mmol/L Tris-HCl, pH 7.6, containing 1 mmol/L adenosine triphosphate, 20 mmol/L phosphocreatine, 0.2 mg/mL creatine phosphokinase, 6.4 mmol/L MgCl2, 1 mmol/L 3-isobutyl-1-methylxanthine, and 0.02 mmol/L guanosine triphosphate). The reaction was then stopped after 15 minutes by application of cold solution consisting of 50 mmol/L sodium acetate (pH 5.0) and was centrifuged at 1000g for 10 minutes at 4°C. cAMP in the supernatant was measured by equilibrated radioimmunoassay. Iodinated 2´-O-monosuccinyl-adenosine 3´,5´-cyclic monophosphate tyrosyl methyl ester (iodine
125–labeled cAMP) was prepared as described by previous investigators.24 Standards or samples were taken up in a final volume of 100 µL of 50 mmol/L sodium acetate buffer (pH 4.8). A 100 µL sample of dilute cAMP antiserum (Calbiochem-Novabiochem, San Diego, CA) and iodine 125–labeled cAMP (10,000 cpm/100 µL) were added and incubated at 4°C for 15 hours. The bound form was separated from the free form by charcoal suspension, and the supernatant was counted in a gamma counter (Packard, Meriden, CT). All samples in one experiment were analyzed in a single assay. Nonspecific binding was <2.0%. The 50% intercept point was at 16.5 ± 0.8 fmol/tube (n = 10). The intraand interassay coefficients of variation were 5.0% ± 1.2% (n = 10) and 9.6% ± 1.9% (n = 10), respectively. Results were expressed as moles of cAMP generated per milligram of protein per minute. Drugs and statistical analysis. Drugs were purchased from Sigma Chemical Co, St Louis, MO, unless stated otherwise. Results are expressed as mean ± SEM. The statistical significance of differences between the groups was determined with an unpaired t test. RESULTS Kidney functional parameters. Table I summarizes the kidney function data. After the treatment with amphotericin, plasma creatinine clearance was decreased and serum creatinine levels were increased. Urinary flow rates were significantly increased, while the urine-toplasma ratio of osmolality and tubular free water reabsorption were decreased. Expression of AQP water channels. The expression of AQP proteins was determined in the cortex, outer medulla, and inner medulla of the kidney. The antiAQP1 antibody recognized 29-kd and 35- to 50-kd bands corresponding to nonglycosylated and glycosylated AQP1, respectively. The anti-AQP2 antibody recognized 29-kd and 35- to 50-kd bands corre-
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Fig 6. Immunoblots of Gsα proteins in the inner medulla. Representative immunoblots and densitometric data are shown. White column indicates controls, shaded column indicates amphotericin-treated. Each column depicts the mean ± SEM of 6 rats. *P < .05 as compared with controls.
sponding to nonglycosylated and glycosylated AQP2, respectively. The anti-AQP3 antibody recognized 27kd and 33- to 40-kd bands corresponding to nonglycosylated and glycosylated AQP3, respectively. After the treatment with amphotericin, the expression of AQP2 was decreased in the outer medulla and inner medulla while not in the cortex (Fig 1). It was decreased in parallel in the membrane-enriched and cytoplasmic fractions (Fig 2). Neither the expression of AQP1 nor that of AQP3 was significantly altered in the cortex, outer medulla, or inner medulla (Figs 3 and 4). Adenylyl cyclase activity. The inner medulla was examined for its adenylyl cyclase activity (Fig 5). After the treatment with amphotericin, the cAMP generation in response to AVP was decreased in the inner medulla. The cAMP generation stimulated by sodium fluoride was also significantly blunted by amphotericin, whereas that provoked by forskolin was not significantly affected. Protein expression of Gsα and adenylyl cyclase VI. Fig 6 shows immunoblots of Gsα proteins expressed in the inner medulla. The anti-Gsα antibody recognized a doublet at 50 kd and at 45 kd. The treatment with amphotericin significantly decreased the expression of Gsα proteins. Fig 7 shows its immunoblots of type VI adenylyl cyclase. The antibody recognized a broad band around 160 kd that was not significantly affected by the amphotericin treatment.
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Fig 7. Immunoblots of type VI adenylyl cyclase in the inner medulla. White column indicates controls, shaded column indicates amphotericin-treated. Each column depicts the mean ± SEM of 6 rats.
Table I. Renal functional parameters in control and amphotericin-treated rats Control (n = 6)
Pcr (mg/dL) Ccr (mL/min) Urinary flow rate (µL/h) Posm (mOsm/kg H2O) Uosm (mOsm/kg H2O) (U/P)osm TcH2O (µL/min/kg)
0.41 1.34 215.6 289.8 1758.4 6.07 69.7
± ± ± ± ± ± ±
0.03 0.15 21.4 9.7 138.4 0.5 14.5
Amphotericin (n = 5)
0.96 0.51 758.7 321.2 627.9 1.95 37.4
± ± ± ± ± ± ±
0.07* 0.12† 103.2* 8.6‡ 56.3† 0.3† 11.7‡
Values are expressed as mean ± SEM. n, Number of rats; Pcr, plasma creatinine; Ccr, creatinine clearance; Posm, plasma osmolality; Uosm, urine osmolality; (U/P)osm, urine-to-plasma ratio of osmolality; TcH2O, solute-free water reabsorption. *P < .01 versus control. †P < .001 versus control. ‡P < .05 versus control.
DISCUSSION
Treatment with amphotericin resulted in a polyuric kidney failure. The serum creatinine level was increased along with a decrease in its renal clearance. Urine osmolality and tubular free water reabsorption were also decreased, indicating an impaired concentrating ability. These findings are in accord with those in patients treated with amphotericin, in which the impaired urinary concentration may represent nephrogenic diabetes insipidus with polyuria and hypotonic urine.5,6 It has also been shown that amphotericin decreases the stimulatory action of the AVP/cAMP
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pathway on water permeability in the inner medullary collecting duct in rats.25 However, the downstream of the AVP/cAMP pathway has not been explored in amphotericin-induced nephropathy. It has been known that the AVP/cAMP pathway has long-term and short-term regulatory effects on AQP2 water channels in the collecting duct.12,13 The present study showed that the total abundance of AQP2 proteins was decreased by amphotericin. The decrease was parallel in the membrane-enriched and cytoplasmic fractions, suggesting a preserved targeting. Similar findings were noted in rats with such acquired forms of nephrogenic diabetes insipidus syndromes as cisplatin-induced nephropathy,20 lithium-induced nephropathy,26 and chronic kidney failure induced by surgical renal mass reduction.19 Nevertheless, it is likely that there may have been an initial transient impairment of AQP2 targeting after amphotericin treatment, while a reduced total abundance may prevail in the long run. The cAMP generation in response to AVP was significantly blunted after treatment with amphotericin. Therefore the primary site of impairment was further determined by the generation of cAMP with different pharmacologic tools. The adenylyl cyclase activity in response to sodium fluoride, which activates the adenylyl cyclase in a receptor-independent but G-protein– dependent manner,27 was attenuated after amphotericin treatment. In contrast, the cAMP generation stimulated by forskolin, which directly activates the catalytic unit of adenylyl cyclase,28 was not affected. These findings indicate that the G protein may be the site primarily injured by amphotericin. The speculation is substantiated by the reduced expression of Gsα proteins, with no significant alterations of adenylyl cyclase VI expression. A primary derangement of G protein may in turn result in a failure to adequately stimulate the catalytic unit of the adenylyl cyclase and hence the generation of cAMP. AQP1 is extremely abundant in the proximal tubule and descending thin limb, and AQP1 gene knockout mice have demonstrated 80% to 90% reduction of osmotic water permeability therein.9,29 It appears to provide the chief route of water reabsorption in the proximal nephron. An important role of AQP3 in urinary concentration has also been suggested by a remarkable polyuria and polydipsia in AQP3-null mice.30 In contrast to the expression of AQP2, however, neither the expression of AQP1 nor that of AQP3 was significantly altered by amphotericin. This finding suggests that AQP1 and AQP3 channels are not involved in the amphotericin-induced impairment of urinary concentration. It also suggests that different regulatory mechanisms exist between AQP1/AQP3 and AQP2.
In summary, the expression of AQP2 water channels was decreased in the kidney after treatment with amphotericin in association with a polyuric kidney failure. The decreased AQP2 expression may at least in part be causally related to the impairment of urinary concentration. On the other hand, the primary impairment in the pathway leading to the activation of AQP channels that are activated by the AVP/cAMP system may lie at the level of G proteins.
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