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Aflatoxicosis alters avian renal function, calcium, and vitamin d metabolism R. P. Glahn b
a e
a
f
, K. W. Beers , W. G. Bottje , R. F. Wideman c
Jr. , W. E. Huff & W. Thomas
d
a
Department of Animal and Poultry Sciences , University of Arkansas , Fayetteville, Arkansas b
Department of Poultry Science , Pennsylvania State University , University Park, Pennsylvania c
U.S. Department of Agriculture , Agriculture Research Service , College Station, Texas d
Department of Pediatrics , Little Rock Medical School , Little Rock, Arkansas e
Mayo Clinic Nephrology Research Unit , Guggenheim Bldg. (9), Rochester, MN, 55905 f
Department of Animal and Poultry Sciences , University of Arkansas , Fayetteville, AR, 72701 Published online: 15 Oct 2009.
To cite this article: R. P. Glahn , K. W. Beers , W. G. Bottje , R. F. Wideman Jr. , W. E. Huff & W. Thomas (1991) Aflatoxicosis alters avian renal function, calcium, and vitamin d metabolism, Journal of Toxicology and Environmental Health, 34:3, 309-321, DOI: 10.1080/15287399109531570 To link to this article: http://dx.doi.org/10.1080/15287399109531570
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AFLATOXICOSIS ALTERS AVIAN RENAL FUNCTION, CALCIUM, AND VITAMIN D METABOLISM R. P. Glahn, K. W. Beers, W. G. Bottje Department of Animal and Poultry Sciences, University of Arkansas, Fayetteville, Arkansas
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R. F. Wideman, Jr. Department of Poultry Science, Pennsylvania State University, University Park, Pennsylvania W. E. Huff U.S. Department of Agriculture, Agriculture Research Service, College Station, Texas W. Thomas Department of Pediatrics, Little Rock Medical School, Little Rock, Arkansas
Experiments were designed to determine the effects of aflatoxicosis on avian renal function, calcium (CA), inorganic phosphorous (P1),and vitamin D metabolism, and to determine if the effects of aflatoxin are reversible upon discontinuation of toxin administration. Three-week-old male broiler chickens (n = 12 per treatment) received aflatoxin (AF; 2 mg/kg po) or an equal volume of corn oil, the AF carrier vehicle, for 10 consecutive days. After 10 d of treatment, half of the birds from each treatment group were anesthetized and prepared for renal function analysis, which included a 2-h phosphate loading period. Ten days after discontinuation of AF treatment, the remaining birds in each treatment group were anesthetized and prepared for renal function analysis. AF decreased plasma 25-hydroxy vitamin D [25(OH)D] and 1,25-dihydroxy vitamin D [1,25(OH)2D] levels after 5 d of treatment. After 10 d of treatment, urine flow rate (V), fractional sodium excretion (FENa), and fractional potassium excretion (FEK) were lower in AF-treated birds. In addition, total plasma Ca tended to be lower
Published with the approval of the Director of the University of Arkansas Agricultural Experiment Station. The authors wish to thank Joyce Satnick for her fine technical assistance. This research was supported in part by R29GM38612 from the National Institute of Health to Dr. Walter Bottje. Current address for R. P. Glahn is Mayo Clinic Nephrology Research Unit, Guggenheim Bldg. (9), Rochester, MN 55905. Requests for reprints should be sent to Walter G. Bottje, Ph.D., Department of Animal and Poultry Sciences, University of Arkansas, Fayetteville, AR 72701.
309 Journal of Toxicology and Environmental Health, 34:309-321, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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(p - .10) and fractional Ca excretion (FECa) tended to be higher (p = .10) in the AFtreated birds. Intravenous phosphate loading produced a sharp increase in urine hydrogen ion concentration ([H+]) in the AF-treated birds. Glomerular filtration rate (CFR) was reduced and plasma osmolality was increased in AF-treated birds 10 d after discontinuation of toxin administration.
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The results indicate that AF directly or indirectly affects Ca and Pi metabolism in avians. At the present time, the effects may be related to altered vitamin D and parathyroid hormone (PTH) metabolism. Aflatoxicosis may decrease endogenous PTH synthesis and the renal sensitivity to PTH. The AF-related increase in urine [H+] during phosphate loading is probably due to increased Na+/H+ counterport, suggesting that AF stimulates sodium reabsorption. Also, the decrease in GFR exhibited 10 d after toxin removal indicates that AF may cause prolonged alteration in renal function.
INTRODUCTION Previous studies have shown that nephrotoxic mycotoxins such as citrinin and ochratoxin A can alter avian renal function and Ca and P, excretion (Hnatow and Wideman, 1985; Glahn et al., 1988b, 1989a). Although primarily a hepatotoxin (Uchida et al., 1988), aflatoxin (AF) has been shown to have nephrotoxic capabilities in domestic fowl. Kidney weight is increased during aflatoxicosis (Tung et al., 1973; Huff et al., 1986a, 1986b; Glahn et al., 1990), as is the ability of the avian kidney to excrete phenol red (Tung et al., 1973). Detailed analysis of avian renal function during aflatoxicosis has shown that AF decreases fractional excretion of phosphorous (FEP) and total plasma Ca (Glahn et al., 1990). These observations indicate that AF alters Ca and P| metabolism in domestic fowl. There are several mechanisms whereby AF may alter Ca and P, metabolism. Aflatoxin may act at the level of the intestine to decrease Ca absorption; it may directly damage renal tissue; it may alter blood levels of parathyroid hormone (PTH); or AF may change the renal response to PTH. In addition, AF may affect hepatic synthesis of 25(OH)D or the renal production of 1,25(OH)2D, thus indirectly altering the renal and parathyroid regulaton of Ca and P,. The objectives of the present study were to obtain an initial assessment of the possible mechanism(s) whereby AF alters renal function and Ca and P, metabolism. In addition, the present study sought to determine if the effects of aflatoxicosis on renal function persist after discontinuation of AF administration. Experiments were designed to measure renal function, plasma 25(OH)D, and plasma 1,25(OH)2D during severe aflatoxicosis and 10 d after cessation of AF administration. Since previous studies have shown that AF-treated birds exhibited decreased FEP, the renal function analysis included a 120-min phosphate loading period, which would challenge the kidney's ability to handle phosphorus. Phos-
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phate loading should increase filtered P, and maximize endogenous PTH release by depressing blood ionized Ca, thus providing maximal phosphaturic stimulus on the kidney (Wideman et al., 1980; Wideman, 1987). Blood levels of PTH were not measured since assay methods for avian PTH have not yet been developed, and mammalian PTH assay kits are inadequate for avian species (Koch et al., 1984). MATERIALS AND METHODS
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Animals
A total of 24 3-wk-old commercial broiler chickens were divided into control and aflatoxin-treated groups (n = 12 per group). The birds were housed in battery cages and had ad libitum access to water and a starter ration (3000 kcal/kg, 20% crude protein). Aflatoxin Production and Administration
For 10 consecutive days prior to renal function analysis, birds receiving aflatoxin were gavaged with an aflatoxin-containing rice powder suspended in 100% corn oil. The concentration of aflatoxin in corn oil was 0.56 mg/ml and was administered at a dose of 2 mg/kg body weight/d. HPLC analysis of the aflatoxin-containing rice powder yielded a total aflatoxin concentration of 1.12 mg/kg rice powder, which consisted of 79,16, 1, and 4% aflatoxin B1; B2, Gv and G2, respectively. Control birds received an equal volume of corn oil, the aflatoxin carrier vehicle. Previous studies had shown that the above dose and mode of administration had profound effects on renal function (Glahn et al., 1990). The aflatoxincontaining rice powder was produced in the laboratory of W. E. Huff, U.S. Department of Agriculture, Agricultural Research Station, College Station, Tex. Methods of aflatoxin production were as previously described (Huff and Doerr, 1981). Surgical Preparation
On d 11 of the experiment (experient 1), half of the birds from each treatment group (n = 6 per group) were anesthetized with intramuscular injections of a combination of diallylbarbituric acid, urethane, and monoethyl urea (DIAL, quarter strength; 2.0 ml/kg body weight; CibaGeigy Pharmaceutical Co., Summit, N.J.) and prepared for renal function analysis. Supplemental injections of DIAL were given as needed to maintain general anesthesia. A carotid artery and a brachial vein were cannulated for blood sampling and systemic intravenous infusion, respectively. An abdominal incision was made and the posterior portion of the colon was tied off to prevent fecal contamination of the urine. Ureteral urine was collected by using cyanoacrylate adhesive to seal a pipet tip to the cloaca of each bird. On d 21 of the experiment (experiment 2), the re-
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maining birds from the control and aflatoxin-treated groups (n = 6 per group) were anesthetized and prepared for renal function analysis as described above. Experimental Protocol for Renal Function Analysis Upon completion of the surgical preparation, a solution containing 2 g/l inulin, 2 g/l para-aminohippuric acid (PAH), and 25 g/l mannitol was infused via the brachial catheter at a rate of 0.1 ml/kg body weight/min. All birds were followed to equilibrate at this infusion rate for 20 min prior to the start of urine collection. Urine was then collected for 3 consecutive 20-min periods (periods 1-3). At the end of period 3, sodium phosphate (100 mM, pH = 7.4) was added to the infusion solution, and the infusion rate was increased to 0.4 ml/kg body weight/min. Urine was then collected for 6 consecutive 20-min periods (periods 4-9). Arterial blood samples (1.5 ml) were taken every 30 min from the start of urine collection. At the end of period 3, an additional 2 ml of arterial blood was collected for measurement of vitamin D metabolites. Upon completion of period 9, the birds were euthanized with an overdose of DIAL and the kidneys were individually weighed and inspected for gross pathology. Sample Handling and Analysis Timed urine samples were collected in preweighed tubes for gravimetric determination of urine volume and flow rate. Equal volumes of urine and 0.5 M LiOH were then mixed in a separate tube to dissolve uric acid precipitates. Urine pH was measured immediately upon collection. Arterial blood samples were collected in heparinized tubes and immediately measured for blood ionized Ca content prior to prompt centrifugation. All urine and blood samples were quickly stored and frozen in airtight containers to prevent dessication, and were thawed immediately prior to analysis. Colorimetric assays were used to measure inulin (Waugh, 1977), PAH (Brun, 1957), and P, (Fiske and Subbarow, 1925). Sodium (Na) and potassium (K) were measured by flame photometry. Total Ca in urine and plasma was measured by atomic absorption. Determination of vitamin D metabolites was as previously described (Hollis and Pittard, 1984). Total plasma protein was determined with a commercial kit (Sigma Chemical Co., St. Louis, Mo.). Calculation of Renal Function Variables Urine samples were converted to urine flow rates (V; ml/kg body weight/min). Glomerular filtration rate (GFR; ml/kg body weight/min) was calculated as the clearance of inulin (urine inulin concentration/plasma inulin concentration x V). The value V/GFR represents the fraction of GFR excreted as urine. Excretion data for ?„ Na, K, and Ca are expressed
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as fractional excretion rates (FE), which were calculated as the clearance of each substance divided by the GFR. Clearance values for PAH were calculated for estimation of renal plasma flow (CPAH, ml/kg BW/min). Filtration fraction (FF) was calculated as GFR/CPAH. Calcium clearance values were calculated using ionized plasma Ca concentrations, as ionized plasma Ca closely matches ultrafilterable Ca. Urine hydrogen ion concentration ([H+]) was calculated from measurement of urine pH, and is expressed as equivalents per liter x10~5. Plasma values for osmolality is expressed as mOsm/kg H2O. Plasma values for Na, K, and Ca are expressed as mH. Plasma P, is expressed as mg/dL Downloaded by [New York University] at 00:39 25 June 2015
Statistical Analysis
Statistical analysis was performed using the SAS general linear models procedure to generate the appropriate analysis of variance tables (SAS Institute, Inc., 1985). Least significant differences (LSD) at a 95% confidence level (p < .05) were then calculated according to the methods of Milliken and Johnson (1984). Values were considered significantly different if the difference between the intergroup means was larger than the LSD. Values that approached significance (.10 > p > .05) were also reported. RESULTS Body Weight, Kidney Weight, Vitamin D, and Plasma Protein Values
In experiment 1, AF decreased average daily body weight and increased kidney weight (Table 1). AF decreased plasma levels of 25(OH)D TABLE 1. Measurements (Mean ± SE) of Body Weight, Average Daily Body Weight Cain, and Kidney Weight in Control and Aflatoxin-Treated Birds Experiment 2
Experiment 1 Variable
Control
Aflatoxin
Control
Aflatoxin
Body weight (kg) Average daily gain (g) Kidney weight (g) gKW/100 gBW3
1.27 64 12.3 0.97
1.25 44 18.3 1.46
1.41 81 15.9 1.12
1.35 84 23.7 1.76
± + ± ±
0.02 4 0.9 0.07
± ± ± ±
0.10 3** 1.7** 0.14**
± 0.14 ± 11 ±1.5 ± 0.11
± 0.05 ± 9 ± 2.1** ± 0.15**
Note. Values are presented at 10 d of AF treatment (experiment 1), and after 10 d of AF treatment followed by a 10-d recovery period (experiment 2). Double asterisk indicates significant difference (p < .05) from control birds. Body weight and kidney weight values are measured on the day of the experiment. Average daily gain values for experiment 1 represent the average daily gain for the 10 d of AF treatment. In experiment 2, the average daily gain values are for the 10 d post AF treatment. a Values are expressed as grams of kidney weight per 100 g body weight.
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and 1,25(OH)2D after 5 d of AF treatment (Table 2). Because of insufficient amount of plasma, 1,25(OH)2D values could not be determined in experiment 2. For experiment 2, average daily gain was not different, but kidney weight remained higher in AF-treated birds. Plasma protein decreased after 5 and 10 d of AF treatment but was not different from that of control birds 10 d after discontinuation of toxin treatment (Table 2). Experiment 1 Prior to phosphate loading, ifV/GFR, FENa, and FEK were lower (p < .05) in AF-treated birds Gable 3). In addition, AF tended to increase FECa and decrease total plasma Ca (p = .10). During phosphate loading, urine [H + ] increased dramatically (Fig. 1) within 60 min and continued to increase throughout the experiment. Although FENa increased in both groups during phosphate loading, FENa remained lower in the AFtreated birds throughout the experiment (Table 3). FEK was lower in AFtreated birds 120 min after the initiation of phosphate loading. Also, total Ca tended to be lower (p = .10) in AF-treated birds after 120 min of phosphate loading. Intergroup differences in V and V/GFR were not maintained during the phosphate loading periods. Infusion of the 100 mM sodium phosphate increased plasma P,, while concomitantly decreasing total Ca and blood ionized Ca2+ (Tables 3 and 4). Experiment 2
On d 20 of the experiment (10 d after cessation of toxin gavage), GFR was lower (p < .05) and plasma osmolality was higher (p < .05) in AFtreated birds (Table 4). No other differences in renal function were observed prior to phosphate loading. During phosphate loading, V tended to be lower (p = .07-.08) in AF-treated birds after 60 and 120 min of phosphate loading. GFR also tended to remain lower (p = .07) in AFtreated birds after 60 min of phosphate loading. Urine [H + ] increased in AF-treated birds after 120 min of phosphate loading (Table 4), thus showing a reduction in the response from experiment 1. FENa increased in both groups during phosphate loading but remained lower in the AFtreated group after 60 min. DISCUSSION
The present study is the first to simultaneously examine vitamin D status and renal function during aflatoxicosis in birds. The results support previous research showing that AF decreases FEP and total plasma Ca, while concomitantly tending to increase FECa (Glahn et al., 1990). In the present study, AF decreased plasma levels of 25(OH)D and 1,25(OH)2D within 5 d of toxin exposure. Similar results have been reported in rats (Sergeev et al., 1988), indicating that AF alters vitamin D metabolism in both birds and mammals. Also, effects on the renal han-
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TABLE 2. Plasma 25(OH)D and 1,25(OH)2D Levels for Control and Aflatoxin-Treated Birds Experiment 2
Experiment 1
5d
25(OH)D (ng/ml) 1,25(OH)2D (pg/ml) Total plasma protein (g/dl)
10 d
10 d + 10 d recovery
Control
Aflatoxin
Control
Aflatoxin
Control
Aflatoxin
15.1 ± 1.6 98.3 ± 15.3 2.9 ± 0.1
10.4 ± 0.7** 39.6 ± 4.4** 2.3 ± 0.1**
15.7 ± 0.6 49.1 ± 2.8 2.1 ± 0.1
12.2 ± 2.1 30.3 ± 2.4** 1.5 ± 0.2**
15.0 ± .1
14.7 ± .2
— 2.2 ± 0.2
— 2.3 ± 0.2
Note. Values are presented at 5 and 10 d of AF treatment (experiment 1), and after 10 d of AF treatment followed by a 10-d recovery period (experiment 2). Double asterisk indicates significant difference (p < .05) from control bird.
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TABLE 3. Renal Function and Plasma Electrolyte Values (Mean ± SE) of Control and Aflaxotin-Treated Birds After 10 Days of Treatment (Experiment 1) 0 min after phosphate loading Values
Control
V
0.047 1.78 0.027 13.1 0.146 0.383 0.544 0.027 0.009 0.499 283 8.1 2.11 1.26 153 4.90
GFR V/GFR CPAH FF Urine [ H + ] FEP FECa FENa FEK Plasma osmolality Plasma Pj Total Ca Blood ion Ca 2+ Plasma Na Plasma K
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.006 0.26 0.002 1.8 0.023 0.104 0.083 0.009 0.002 0.045 2 0.6 0.12 0.06 3 0.31
60 min after phosphate loading
120 min after phosphate loading
Aflatoxin
Control
Aflatoxin
Control
0.029 2.20 0.015 16.9 0.135 0.312 0.516 0.108 0.002 0.192 285 8.1 1.80 1.23 156 4.47
0.099 2.57 0.039 27.3 0.098 0.424 0.739 0.016 0.016 0.566 278 16.6 1.56 0.71 160 4.90
0.095 2.75 0.036 32.1 0.088 1.598 0.597 0.104 0.007 0.451 287 19.5 1.23 0.63 157 4.48
0.109 2.40 0.044 29.7 0.084 0.501 0.732 0.062 0.019 0.761 281 20.3 1.24 0.52 159 3.75
± 0.002** ± 0.36 ± 0.002** ± 2.0 ± 0.019 ± 0.075 ± 0.122 ± 0.048* ± 0.001** ± 0.032** ±4 ± 0.3 ± 0.16* ± 0.25 ± 4 ± 0.28
± 0.014' ± 0.35' ± 0.002' ± 3.4' ± 0.012 ±0.115 ± 0.128 ± 0.006 ± 0.003' ± 0.085 ± 3 ± 1.5' ± 0.29' ± 0.06' ± 4 ± 0.24
± 0.006' ± 0.27 ± 0.003' ± 3.3' ± 0.008 ± 0.421**' ± 0.138 ± 0.038* ± 0.001**' ± 0.072' ± 7 ± 1.2' ± 0.25' ± 0.03' ±5 ± 0.44
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Aflatoxin 0.023' 0.32 0.004' 4.7' 0.010 0.103 0.150 0.039 0.003' 0.085' 1 2.4' 0.26' 0.03' 6 0.20'
0.099 3.04 0.034 32.9 0.092 2.549 0.493 0.052 0.010 0.476 285 23.0 0.70 0.50 166 3.78
± 0.007' ± 0.32' ± 0.004' ± 4.1' ± 0.010 ± 0.605**' ± 0.101 ± 0.018 ± 0.002**' ± 0.065**' ±2 ± 1.5' ± 0.22*' ± 0.06' ± 3' ± 0.19'
Note. Single asterisk indicates intergroup difference (p < .10) at the designated timepoint. Double asterisk indicates intergroup difference (p < .05) S) at the designated timepoint. 'indicates intragroup difference (p < .05) from prephosphate loading period).
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7
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K 0) s-
-60
-40
80
100
120
Time (min) FIGURE T. Urine hydrogen ion concentration ([H + ]) during experiment 1 (i.e., after 10 d of aflatoxin treatment). Asterisk indicates significant difference (p < .05) from the control group.
dling of Ca and P, similar to those observed in the present study have been produced by decreased levels of PTH (Wideman, 1987; Glahn et al., 1988a), and hypocalcemia can be induced via vitamin D deficiency (Forte et al., 1982). The observed effects of AF on Ca and P, metabolism may reflect decreased efficacy or circulating titers of PTH (not measured) in conjunction with significant alterations in vitamin D status (Wideman, 1987). Unfortunately, an assay for avian PTH is not available (W. Burke, personal communication), and the effects of AF on PTH, Ca, and P, metabolism in a mammalian model have not been reported. Avian kidneys are both an endocrine source and a target tissue for vitamin D (Wideman, 1987). Hypocalcemia stimulates PTH secretion, which in turn stimulates renal adenylate cyclase, activating 25hydroxycholecalciferol 1a-hydroxylase to increase 1,25(OH)2D synthesis (Rasmussen et al., 1972; Fraser and Kodicek, 1973). As 1,25(OH)2D and 25(OH)D levels were decreased in the present study, it is likely that hepatic production of 25(OH)D was inhibited by AF. These observations also indicate that AF may inhibit PTH secretion and/or renal synthesis of 1,25(OH)2D. AF also may decrease the renal sensitivity to PTH and vitamin D. Interestingly, decreased renal sensitivity to PTH has been observed in vitamin D-deficient chicks (Forte et al., 1982). Vitamin D metabolites have not been shown to have direct effects on
u
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03
TABLE 4. Renal Function and Plasma Electrolyte Values (Mean ± SE) of Control and Aflatoxin-Treated Birds for Experiment 2 (i.e., 10 Days Post 10 Days of Aflatoxin or Corn Oil Treatment) 0 min after phosphate loading Values
Control
V CFR V/CFR CPAH FF Urine [ H + ] FEP FECa FENa FEK Plasma osmolality Plasma P| Total plasma Ca Blood ion Ca 2 + Plasma Na Plasma K
0.039 01.84 0.022 10.3 0.201 0.911 0.805 0.035 0.007 0.214 277 6.18 2.54 1.23 156 4.41
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.004 0.08 0.002 1.4 0.024 0.105 0.218 0.010 0.002 0.024 4 0.24 0.08 0.14 6 0.31
60 min after phosphate loading
Aflatoxin
Control
0.034 1.47 0.024 7.67 0.222 0.892 0.400 0.035 0.003 0.259 292 6.02 2.50 1.00 161 4.73
0.112 2.38 0.048 19.8 0.125 0.593 0.518 0.081 0.026 0.393 284 16.95 1.70 0.58 161 5.50
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.002 0.12** 0.002 1.3 0.037 0.159 0.176 0.008 0.001 0.048 2** 0.19 0.04 0.17 7 0.32
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.008' 0.10 0.004' 2.1' 0.011' 0.112 0.357 0.024 0.002' 0.063' 4 1.14' 0.08' 0.05' 9 1.24
120 min after phosphate loading
Aflatoxin
Control
0.089 ± 0.018*' 1.86 ± 0.40* 0.047 ± 0.003' 21.9 ± 4.7f 0.088 ± 0.010' 1.003 ± 0.218 0.541 ± 0.370 0.071 ± 0.030 0.017 ± 0.003**' 0.536 ± 0.131' 280 ± 2 15.62 ± 0.25' 2.22 ± 0.25 0.73 ± 0.07 161 ± 7 4.60 ± 0.40
0.113 2.08 0.056 24.6 0.105 0.538 0.783 0.111 0.031 0.564 291 21.10 1.24 0.43 160 3.90
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Aflatoxin 0.006' 0.19 0.006' 7.2' 0.025' 0.161 0.504 0.039 0.003' 0.074' 5' 1.73' 0.11' 0.05' 11 0.35
0.092 1.80 0.054 33.4 0.071 1.553 0.609 0.125 0.017 0.570 282 19.04 1.32 0.49 164 4.52
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.005*' 0.26 0.007' 9' 0.018' 0.528**' 0.333 0.035 0.004**' 0.104' 4' 0.92' 0.26' 0.05' 8 0.44
Note. Single asterisk indicates intergroup difference (p < .10) at the designated timepoint. Double asterisk indicates intergroup difference (p < .05) at the designated timepoint. 'indicates intragroup difference (p < .05) from prephosphate loading period).
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the renal handling of Ca in the avian kidney (Wideman, 1987); however, 1,25(OH)2D has been shown to stimulate the synthesis of a specific calcium-binding protein in avian distal tubules and collecting ducts (Narbaitz et al., 1982). Thus, 1,25(OH)2D may contribute to distal tubular finetuning of Ca excretion, and inhibition of this process by AF could account for some of the increase in FECa observed during aflatoxicosis. Aflatoxin inhibits DNA-dependent RNA polymerase and causes impairment of nucleolar DNA template function, resulting in general inhibition of protein synthesis (Gelboin et al., 1966; Yu, 1977). As a result, hypoproteinemia is a common effect of aflatoxicosis (Huff et al., 1986a, 1986b). In the present study, total plasma protein and blood ionized Ca were measured to determine if AF-induced hypoproteinemia alters blood ionized Ca, which in turn would affect circulating levels of PTH (Mueller et al., 1970). Blood ionized Ca was not altered in AF-treated animals even though total plasma protein was reduced, indicating the observed changes in the renal handling of Ca and Pi may not be due to changes in PTH synthesis induced by ionized Ca. Alternatively, AF may inhibit DNA template function in an endocrine organ such as the parathyroid gland, thereby reducing hormone synthesis and indirectly causing the observed effects on Ca and P, metabolism. In avians, phosphate accounts for approximately 25-33% of the urinary buffering capacity (Craan et al., 1982; Long and Skadhauge, 1983). At normal pH values, filtered phosphate is in the dibasic form (HPO42~). Urinary acidification titrates dibasic phosphate to its monobasic form (H2PO4~), which is excreted in the urine. In the present study, the increase in urine [H + ] during phosphate loading may reflect increased Na + /H + counterport. This effect on urine [H + ] may be secondary to an AF-induced increased in Na reabsorption, as evident by decreased FENa and increased plasma Na within the AF-treated birds of the present study (Table 3). Infusion of the 100 mM sodium phosphate provided the necessary Na and phosphate to stimulate additional Na + /H + counterport. It is interesting to note that indicators of diuresis and diarrhea, such as wet manure and increased urine flow rate, were not observed in the AFtreated birds. Therefore, increased Na reabsorption in the present study was not a secondary response to toxin-induced fluid or electrolyte loss via the kidneys or intestine. Ten days after cessation of toxin exposure, previously observed differences in V, V/GFR, FENa, FEK, and total plasma Ca were absent prior to phosphate loading (Tables 3 and 4). Also, the AF-induced effect on urine [H + ] was attenuated during the phosphate loading periods (Table 4). These results suggest that AF-related effects on the above parameters are reversible upon cessation of AF exposure. However, prior to phosphate loading GFR was decreased and plasma osmolality was increased in the AF-treated binds 10 d after AF administration was discontinued (Table 4). These differences, which were not present in experiment 1, indicate that
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AF may have prolonged effects on renal function. Decreased GFR can reflect a reduction in the functional renal mass of avian kidneys (Niznik et al., 1985; Glahn et al., 1989b). In summary, AF directly or indirectly affects Ca and P, metabolism in avians. At the present time, these effects may be related to altered vitamin D metabolism. Aflatoxicosis may also decrease endogenous PTH synthesis and decrease renal sensitivity to PTH. Alternatively, AF could extensively inhibit DNA template function such that endocrine organs such as the liver, kidney, parathyroid gland, and so forth exhibit altered sensitivity and secretion. REFERENCES Brun, C. 1957. A rapid method for determination of para-aminohippuric acid in kidney function tests. J. Lab. Clin Med. 37:955-958. Craan, A. G., Lemieux, G., Vinay, P., and Gougoux, A. 1982. The kidney of the chicken adapts. Chronic metabolic acidosis: In vivo and in vitro studies. Kidney Int. 22:103-111. Fiske, C. H., and Subbarow, Y. 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66:375-400. Forte, L. R., Langeluttig, S. G., Poelling, R. E., and Thomas, M. L. 1982. Renal parathyroid hormone receptors in the chick: Downregulation in secondary hyperparathyroid animal models. Am. J. Physiol. 242:E154-E163. Fraser, D. R., and Kodicek, E. 1973. Regulation of 25-hydroxycholecalciferol 1-hydroxylase activity in kidney by parathyroid hormone. Nature New Biol. 241:163-166. Gelboin, H. V., Wortham, J. S., Wilson, R. G., Friedman, M., and Wogan, G. N. 1966. Rapid and marked inhibition of rat-liver RNA polymerase by aflatoxin B1. Science 154:1205-1206. Glahn, R. P., Wideman, R. F., Jr. and Cowen, B. S. 1988a. Effect of gray strain infectious bronchitis virus and high dietary calcium on renal function of single comb white leghorn pullets at 6, 10, and 18 weeks of age. Poult. Sci. 67:1250-1263. Glahn, R. P., Wideman, R. F., Jr., Evangelisti, J. W., and Huff, W. E. 1988b. Effects of ochratoxin A alone and in combination with citrinin on kidney function of single comb white leghorn pullets. Poult. Sci. 67:1034-1042. Glahn, R. P., Shapiro, R. S., Vena, V. E., Wideman, R. F., Jr., and Huff, W. E. 1989a. Effects of chronic ochratoxin A and citrinin toxicosis on kidney function of single comb white leghorn pullets. Poult. Sci. 68:1205-1212. Glahn, R. P., Wideman, R. F., Jr., and Cowen, B. S. 1989b. Order of exposure to high dietary calcium and gray strain infectious bronchitis virus alters real function and the incidence of urolithiasis. Poult. Sci. 68:1193-1204. Glahn, R. P., Beers, K. W., Bottje, W. G., Wideman, R. F., Jr., and Huff, W. E. 1990. Altered renal function in broilers during aflatoxicosis. Poult. Sci. 69:1796-1797. Hnatow, L. L., and Wideman, R. F., Jr. 1985. Kidney function of single comb white leghorn pullets following acute renal portal infusion of the mycotoxin citrinin. Poult. Sci. 64:1553-1561. Hollis, B. W., and Pittard, W. B. 1984. Relative concentrations of 25-hydroxyvitamin D2/D3 dihydroxyvitamin D2/D3 in maternal plasma at delivery. Nutr. Res. 4:27. Huff, W. E., Kubena, L. F., Harvey, R. B., Hagler, W. M., Jr., Swanson, S. P., Phillips, T. D., and Creger, C. R. 1986a. Individual and combined effects of aflatoxin and deoxynivalenol (DON) in broiler chickens. Poult. Sci. 65:1291-1298. Huff, W. E., Kubena, L. F., Harvey, R. B., Corrier, D. E., and Mollenhauer, H. H. 1986b. Progression of aflatoxicosis in broiler chickens. Poult. Sci. 65:1891-1899. Huff, W. E., and Doerr, J. A. 1981. Synergism between aflatoxin and ochratoxin A in broiler chickens. Poult. Sci. 60:550-555.
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Koch, J., Wideman, R.F.,Jr., and Buss, E. G. 1984. Evaluation of commercial mammalian parathyroid hormone (PTH) radioimmunoassay systems for measuring avian PTH. Comp. Biochem. Physiol. Long, S., and Skadhauge, E. 1983. Renal acid excretion in the domestic fowl. J. Exp. Biol. 104:51. Milliken, G. A., and Johnson, D. E. 1984. Analysis of Messy Data. Vol. 1. Designed Experiments, Chapter 26. Reinhold, N.Y.: Van Nostrand. Mueller, G. L., Constantine, S. A., and Breitenbach, R. P. 1970. Dietary calcium and ultimobranchial body and parathyroid gland in the chicken. Am. J. Physiol. 218(6):1718-1722. Narbaitz, R., Stumpf, W. E., Sar, M., and DeLuca, H. F. 1982. The distal nephron in the chick embryo as a target tissue for 1-alpha-25-dihydroxycholecalciferol. Acta Anat. 112:208-216. Niznik, R. A., Wideman, R. F., Jr., Cowen, B. S., and Kissell, R. E. 1985. Induction of urolithiasis in single comb white leghorn pullets: Effect on glomerular number. Poult. Sri. 64:1430-1437. Rasmussen, H., Wong, M., Bikle, D., and Goodman, D. P. B. 1972. Hormonal control of the renal conversion of 25-hydroxycholecalciferol to 1,25-dihydroxy-cholecalciferol. J. Clin. Invest. 51:2502-2504. SAS Institute, Inc. 1985. SAS User's Guide: Version 5. Cary, N.C.: SAS Institute, Inc. Sergeev, I. N., Arkhapchev, I. P., Kravchenko, L. V., Kodentsova, V. M., and Piliia, N. M. 1988. Effect of mycotoxins aflatoxin B1 and T-2 toxin on the vitamin D3 metabolism and binding of its hormonal form 1,25 dihydroxyvitamin D3 in rats. Vopr. Med. Khim. 34(4):51-57. Tung, H. T., Wyatt, R. D., Thaxton, P., and Hamilton, P. B. 1973. Impairment of kidney function during aflatoxicosis. Poult. Sci. 52:873-878. Uchida, T., Suzuki, K., Esumi, M., Arji, M., and Shikata, T. 1988. Influence of aflation B1 intoxication on duck livers with duck hepatitis B virus infection. Cancer Res. 48:1559-1565. Waugh, W. W. 1977. Photometry in inulin and polyfructosan by use of a cysteine/tryptophan reaction. Clin. Chem. 23:639-645. Wideman, R. F., Jr. 1987. Renal regulation of avian calcium and phosphorous metabolism. J. Nutr. 117:808-815. Wideman, R. F., Jr., Clark, N. B., and Braun, E. J. 1980. Effects of phosphate loading and parathyroid hormone on starling renal phosphate excretion. Am. J. Physiol. 239:F233-F243. Yu, S. 1977. Mechanism of aflatoxin B1 inhibition of rat hepatic nuclear RNA synthesis. J. Biol. Chem. 252:3245-3251. Received January 30, 1991 Accepted June 2, 1991
Journal of Toxicology and Environmental Health Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Aflatoxicosis alters avian renal function, calcium, and vitamin d metabolism R. P. Glahn b
a e
a
f
, K. W. Beers , W. G. Bottje , R. F. Wideman c
Jr. , W. E. Huff & W. Thomas
d
a
Department of Animal and Poultry Sciences , University of Arkansas , Fayetteville, Arkansas b
Department of Poultry Science , Pennsylvania State University , University Park, Pennsylvania c
U.S. Department of Agriculture , Agriculture Research Service , College Station, Texas d
Department of Pediatrics , Little Rock Medical School , Little Rock, Arkansas e
Mayo Clinic Nephrology Research Unit , Guggenheim Bldg. (9), Rochester, MN, 55905 f
Department of Animal and Poultry Sciences , University of Arkansas , Fayetteville, AR, 72701 Published online: 15 Oct 2009.
To cite this article: R. P. Glahn , K. W. Beers , W. G. Bottje , R. F. Wideman Jr. , W. E. Huff & W. Thomas (1991) Aflatoxicosis alters avian renal function, calcium, and vitamin d metabolism, Journal of Toxicology and Environmental Health, 34:3, 309-321, DOI: 10.1080/15287399109531570 To link to this article: http://dx.doi.org/10.1080/15287399109531570
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AFLATOXICOSIS ALTERS AVIAN RENAL FUNCTION, CALCIUM, AND VITAMIN D METABOLISM R. P. Glahn, K. W. Beers, W. G. Bottje Department of Animal and Poultry Sciences, University of Arkansas, Fayetteville, Arkansas
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R. F. Wideman, Jr. Department of Poultry Science, Pennsylvania State University, University Park, Pennsylvania W. E. Huff U.S. Department of Agriculture, Agriculture Research Service, College Station, Texas W. Thomas Department of Pediatrics, Little Rock Medical School, Little Rock, Arkansas
Experiments were designed to determine the effects of aflatoxicosis on avian renal function, calcium (CA), inorganic phosphorous (P1),and vitamin D metabolism, and to determine if the effects of aflatoxin are reversible upon discontinuation of toxin administration. Three-week-old male broiler chickens (n = 12 per treatment) received aflatoxin (AF; 2 mg/kg po) or an equal volume of corn oil, the AF carrier vehicle, for 10 consecutive days. After 10 d of treatment, half of the birds from each treatment group were anesthetized and prepared for renal function analysis, which included a 2-h phosphate loading period. Ten days after discontinuation of AF treatment, the remaining birds in each treatment group were anesthetized and prepared for renal function analysis. AF decreased plasma 25-hydroxy vitamin D [25(OH)D] and 1,25-dihydroxy vitamin D [1,25(OH)2D] levels after 5 d of treatment. After 10 d of treatment, urine flow rate (V), fractional sodium excretion (FENa), and fractional potassium excretion (FEK) were lower in AF-treated birds. In addition, total plasma Ca tended to be lower
Published with the approval of the Director of the University of Arkansas Agricultural Experiment Station. The authors wish to thank Joyce Satnick for her fine technical assistance. This research was supported in part by R29GM38612 from the National Institute of Health to Dr. Walter Bottje. Current address for R. P. Glahn is Mayo Clinic Nephrology Research Unit, Guggenheim Bldg. (9), Rochester, MN 55905. Requests for reprints should be sent to Walter G. Bottje, Ph.D., Department of Animal and Poultry Sciences, University of Arkansas, Fayetteville, AR 72701.
309 Journal of Toxicology and Environmental Health, 34:309-321, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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(p - .10) and fractional Ca excretion (FECa) tended to be higher (p = .10) in the AFtreated birds. Intravenous phosphate loading produced a sharp increase in urine hydrogen ion concentration ([H+]) in the AF-treated birds. Glomerular filtration rate (CFR) was reduced and plasma osmolality was increased in AF-treated birds 10 d after discontinuation of toxin administration.
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The results indicate that AF directly or indirectly affects Ca and Pi metabolism in avians. At the present time, the effects may be related to altered vitamin D and parathyroid hormone (PTH) metabolism. Aflatoxicosis may decrease endogenous PTH synthesis and the renal sensitivity to PTH. The AF-related increase in urine [H+] during phosphate loading is probably due to increased Na+/H+ counterport, suggesting that AF stimulates sodium reabsorption. Also, the decrease in GFR exhibited 10 d after toxin removal indicates that AF may cause prolonged alteration in renal function.
INTRODUCTION Previous studies have shown that nephrotoxic mycotoxins such as citrinin and ochratoxin A can alter avian renal function and Ca and P, excretion (Hnatow and Wideman, 1985; Glahn et al., 1988b, 1989a). Although primarily a hepatotoxin (Uchida et al., 1988), aflatoxin (AF) has been shown to have nephrotoxic capabilities in domestic fowl. Kidney weight is increased during aflatoxicosis (Tung et al., 1973; Huff et al., 1986a, 1986b; Glahn et al., 1990), as is the ability of the avian kidney to excrete phenol red (Tung et al., 1973). Detailed analysis of avian renal function during aflatoxicosis has shown that AF decreases fractional excretion of phosphorous (FEP) and total plasma Ca (Glahn et al., 1990). These observations indicate that AF alters Ca and P| metabolism in domestic fowl. There are several mechanisms whereby AF may alter Ca and P, metabolism. Aflatoxin may act at the level of the intestine to decrease Ca absorption; it may directly damage renal tissue; it may alter blood levels of parathyroid hormone (PTH); or AF may change the renal response to PTH. In addition, AF may affect hepatic synthesis of 25(OH)D or the renal production of 1,25(OH)2D, thus indirectly altering the renal and parathyroid regulaton of Ca and P,. The objectives of the present study were to obtain an initial assessment of the possible mechanism(s) whereby AF alters renal function and Ca and P, metabolism. In addition, the present study sought to determine if the effects of aflatoxicosis on renal function persist after discontinuation of AF administration. Experiments were designed to measure renal function, plasma 25(OH)D, and plasma 1,25(OH)2D during severe aflatoxicosis and 10 d after cessation of AF administration. Since previous studies have shown that AF-treated birds exhibited decreased FEP, the renal function analysis included a 120-min phosphate loading period, which would challenge the kidney's ability to handle phosphorus. Phos-
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phate loading should increase filtered P, and maximize endogenous PTH release by depressing blood ionized Ca, thus providing maximal phosphaturic stimulus on the kidney (Wideman et al., 1980; Wideman, 1987). Blood levels of PTH were not measured since assay methods for avian PTH have not yet been developed, and mammalian PTH assay kits are inadequate for avian species (Koch et al., 1984). MATERIALS AND METHODS
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Animals
A total of 24 3-wk-old commercial broiler chickens were divided into control and aflatoxin-treated groups (n = 12 per group). The birds were housed in battery cages and had ad libitum access to water and a starter ration (3000 kcal/kg, 20% crude protein). Aflatoxin Production and Administration
For 10 consecutive days prior to renal function analysis, birds receiving aflatoxin were gavaged with an aflatoxin-containing rice powder suspended in 100% corn oil. The concentration of aflatoxin in corn oil was 0.56 mg/ml and was administered at a dose of 2 mg/kg body weight/d. HPLC analysis of the aflatoxin-containing rice powder yielded a total aflatoxin concentration of 1.12 mg/kg rice powder, which consisted of 79,16, 1, and 4% aflatoxin B1; B2, Gv and G2, respectively. Control birds received an equal volume of corn oil, the aflatoxin carrier vehicle. Previous studies had shown that the above dose and mode of administration had profound effects on renal function (Glahn et al., 1990). The aflatoxincontaining rice powder was produced in the laboratory of W. E. Huff, U.S. Department of Agriculture, Agricultural Research Station, College Station, Tex. Methods of aflatoxin production were as previously described (Huff and Doerr, 1981). Surgical Preparation
On d 11 of the experiment (experient 1), half of the birds from each treatment group (n = 6 per group) were anesthetized with intramuscular injections of a combination of diallylbarbituric acid, urethane, and monoethyl urea (DIAL, quarter strength; 2.0 ml/kg body weight; CibaGeigy Pharmaceutical Co., Summit, N.J.) and prepared for renal function analysis. Supplemental injections of DIAL were given as needed to maintain general anesthesia. A carotid artery and a brachial vein were cannulated for blood sampling and systemic intravenous infusion, respectively. An abdominal incision was made and the posterior portion of the colon was tied off to prevent fecal contamination of the urine. Ureteral urine was collected by using cyanoacrylate adhesive to seal a pipet tip to the cloaca of each bird. On d 21 of the experiment (experiment 2), the re-
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maining birds from the control and aflatoxin-treated groups (n = 6 per group) were anesthetized and prepared for renal function analysis as described above. Experimental Protocol for Renal Function Analysis Upon completion of the surgical preparation, a solution containing 2 g/l inulin, 2 g/l para-aminohippuric acid (PAH), and 25 g/l mannitol was infused via the brachial catheter at a rate of 0.1 ml/kg body weight/min. All birds were followed to equilibrate at this infusion rate for 20 min prior to the start of urine collection. Urine was then collected for 3 consecutive 20-min periods (periods 1-3). At the end of period 3, sodium phosphate (100 mM, pH = 7.4) was added to the infusion solution, and the infusion rate was increased to 0.4 ml/kg body weight/min. Urine was then collected for 6 consecutive 20-min periods (periods 4-9). Arterial blood samples (1.5 ml) were taken every 30 min from the start of urine collection. At the end of period 3, an additional 2 ml of arterial blood was collected for measurement of vitamin D metabolites. Upon completion of period 9, the birds were euthanized with an overdose of DIAL and the kidneys were individually weighed and inspected for gross pathology. Sample Handling and Analysis Timed urine samples were collected in preweighed tubes for gravimetric determination of urine volume and flow rate. Equal volumes of urine and 0.5 M LiOH were then mixed in a separate tube to dissolve uric acid precipitates. Urine pH was measured immediately upon collection. Arterial blood samples were collected in heparinized tubes and immediately measured for blood ionized Ca content prior to prompt centrifugation. All urine and blood samples were quickly stored and frozen in airtight containers to prevent dessication, and were thawed immediately prior to analysis. Colorimetric assays were used to measure inulin (Waugh, 1977), PAH (Brun, 1957), and P, (Fiske and Subbarow, 1925). Sodium (Na) and potassium (K) were measured by flame photometry. Total Ca in urine and plasma was measured by atomic absorption. Determination of vitamin D metabolites was as previously described (Hollis and Pittard, 1984). Total plasma protein was determined with a commercial kit (Sigma Chemical Co., St. Louis, Mo.). Calculation of Renal Function Variables Urine samples were converted to urine flow rates (V; ml/kg body weight/min). Glomerular filtration rate (GFR; ml/kg body weight/min) was calculated as the clearance of inulin (urine inulin concentration/plasma inulin concentration x V). The value V/GFR represents the fraction of GFR excreted as urine. Excretion data for ?„ Na, K, and Ca are expressed
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as fractional excretion rates (FE), which were calculated as the clearance of each substance divided by the GFR. Clearance values for PAH were calculated for estimation of renal plasma flow (CPAH, ml/kg BW/min). Filtration fraction (FF) was calculated as GFR/CPAH. Calcium clearance values were calculated using ionized plasma Ca concentrations, as ionized plasma Ca closely matches ultrafilterable Ca. Urine hydrogen ion concentration ([H+]) was calculated from measurement of urine pH, and is expressed as equivalents per liter x10~5. Plasma values for osmolality is expressed as mOsm/kg H2O. Plasma values for Na, K, and Ca are expressed as mH. Plasma P, is expressed as mg/dL Downloaded by [New York University] at 00:39 25 June 2015
Statistical Analysis
Statistical analysis was performed using the SAS general linear models procedure to generate the appropriate analysis of variance tables (SAS Institute, Inc., 1985). Least significant differences (LSD) at a 95% confidence level (p < .05) were then calculated according to the methods of Milliken and Johnson (1984). Values were considered significantly different if the difference between the intergroup means was larger than the LSD. Values that approached significance (.10 > p > .05) were also reported. RESULTS Body Weight, Kidney Weight, Vitamin D, and Plasma Protein Values
In experiment 1, AF decreased average daily body weight and increased kidney weight (Table 1). AF decreased plasma levels of 25(OH)D TABLE 1. Measurements (Mean ± SE) of Body Weight, Average Daily Body Weight Cain, and Kidney Weight in Control and Aflatoxin-Treated Birds Experiment 2
Experiment 1 Variable
Control
Aflatoxin
Control
Aflatoxin
Body weight (kg) Average daily gain (g) Kidney weight (g) gKW/100 gBW3
1.27 64 12.3 0.97
1.25 44 18.3 1.46
1.41 81 15.9 1.12
1.35 84 23.7 1.76
± + ± ±
0.02 4 0.9 0.07
± ± ± ±
0.10 3** 1.7** 0.14**
± 0.14 ± 11 ±1.5 ± 0.11
± 0.05 ± 9 ± 2.1** ± 0.15**
Note. Values are presented at 10 d of AF treatment (experiment 1), and after 10 d of AF treatment followed by a 10-d recovery period (experiment 2). Double asterisk indicates significant difference (p < .05) from control birds. Body weight and kidney weight values are measured on the day of the experiment. Average daily gain values for experiment 1 represent the average daily gain for the 10 d of AF treatment. In experiment 2, the average daily gain values are for the 10 d post AF treatment. a Values are expressed as grams of kidney weight per 100 g body weight.
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and 1,25(OH)2D after 5 d of AF treatment (Table 2). Because of insufficient amount of plasma, 1,25(OH)2D values could not be determined in experiment 2. For experiment 2, average daily gain was not different, but kidney weight remained higher in AF-treated birds. Plasma protein decreased after 5 and 10 d of AF treatment but was not different from that of control birds 10 d after discontinuation of toxin treatment (Table 2). Experiment 1 Prior to phosphate loading, ifV/GFR, FENa, and FEK were lower (p < .05) in AF-treated birds Gable 3). In addition, AF tended to increase FECa and decrease total plasma Ca (p = .10). During phosphate loading, urine [H + ] increased dramatically (Fig. 1) within 60 min and continued to increase throughout the experiment. Although FENa increased in both groups during phosphate loading, FENa remained lower in the AFtreated birds throughout the experiment (Table 3). FEK was lower in AFtreated birds 120 min after the initiation of phosphate loading. Also, total Ca tended to be lower (p = .10) in AF-treated birds after 120 min of phosphate loading. Intergroup differences in V and V/GFR were not maintained during the phosphate loading periods. Infusion of the 100 mM sodium phosphate increased plasma P,, while concomitantly decreasing total Ca and blood ionized Ca2+ (Tables 3 and 4). Experiment 2
On d 20 of the experiment (10 d after cessation of toxin gavage), GFR was lower (p < .05) and plasma osmolality was higher (p < .05) in AFtreated birds (Table 4). No other differences in renal function were observed prior to phosphate loading. During phosphate loading, V tended to be lower (p = .07-.08) in AF-treated birds after 60 and 120 min of phosphate loading. GFR also tended to remain lower (p = .07) in AFtreated birds after 60 min of phosphate loading. Urine [H + ] increased in AF-treated birds after 120 min of phosphate loading (Table 4), thus showing a reduction in the response from experiment 1. FENa increased in both groups during phosphate loading but remained lower in the AFtreated group after 60 min. DISCUSSION
The present study is the first to simultaneously examine vitamin D status and renal function during aflatoxicosis in birds. The results support previous research showing that AF decreases FEP and total plasma Ca, while concomitantly tending to increase FECa (Glahn et al., 1990). In the present study, AF decreased plasma levels of 25(OH)D and 1,25(OH)2D within 5 d of toxin exposure. Similar results have been reported in rats (Sergeev et al., 1988), indicating that AF alters vitamin D metabolism in both birds and mammals. Also, effects on the renal han-
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TABLE 2. Plasma 25(OH)D and 1,25(OH)2D Levels for Control and Aflatoxin-Treated Birds Experiment 2
Experiment 1
5d
25(OH)D (ng/ml) 1,25(OH)2D (pg/ml) Total plasma protein (g/dl)
10 d
10 d + 10 d recovery
Control
Aflatoxin
Control
Aflatoxin
Control
Aflatoxin
15.1 ± 1.6 98.3 ± 15.3 2.9 ± 0.1
10.4 ± 0.7** 39.6 ± 4.4** 2.3 ± 0.1**
15.7 ± 0.6 49.1 ± 2.8 2.1 ± 0.1
12.2 ± 2.1 30.3 ± 2.4** 1.5 ± 0.2**
15.0 ± .1
14.7 ± .2
— 2.2 ± 0.2
— 2.3 ± 0.2
Note. Values are presented at 5 and 10 d of AF treatment (experiment 1), and after 10 d of AF treatment followed by a 10-d recovery period (experiment 2). Double asterisk indicates significant difference (p < .05) from control bird.
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TABLE 3. Renal Function and Plasma Electrolyte Values (Mean ± SE) of Control and Aflaxotin-Treated Birds After 10 Days of Treatment (Experiment 1) 0 min after phosphate loading Values
Control
V
0.047 1.78 0.027 13.1 0.146 0.383 0.544 0.027 0.009 0.499 283 8.1 2.11 1.26 153 4.90
GFR V/GFR CPAH FF Urine [ H + ] FEP FECa FENa FEK Plasma osmolality Plasma Pj Total Ca Blood ion Ca 2+ Plasma Na Plasma K
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.006 0.26 0.002 1.8 0.023 0.104 0.083 0.009 0.002 0.045 2 0.6 0.12 0.06 3 0.31
60 min after phosphate loading
120 min after phosphate loading
Aflatoxin
Control
Aflatoxin
Control
0.029 2.20 0.015 16.9 0.135 0.312 0.516 0.108 0.002 0.192 285 8.1 1.80 1.23 156 4.47
0.099 2.57 0.039 27.3 0.098 0.424 0.739 0.016 0.016 0.566 278 16.6 1.56 0.71 160 4.90
0.095 2.75 0.036 32.1 0.088 1.598 0.597 0.104 0.007 0.451 287 19.5 1.23 0.63 157 4.48
0.109 2.40 0.044 29.7 0.084 0.501 0.732 0.062 0.019 0.761 281 20.3 1.24 0.52 159 3.75
± 0.002** ± 0.36 ± 0.002** ± 2.0 ± 0.019 ± 0.075 ± 0.122 ± 0.048* ± 0.001** ± 0.032** ±4 ± 0.3 ± 0.16* ± 0.25 ± 4 ± 0.28
± 0.014' ± 0.35' ± 0.002' ± 3.4' ± 0.012 ±0.115 ± 0.128 ± 0.006 ± 0.003' ± 0.085 ± 3 ± 1.5' ± 0.29' ± 0.06' ± 4 ± 0.24
± 0.006' ± 0.27 ± 0.003' ± 3.3' ± 0.008 ± 0.421**' ± 0.138 ± 0.038* ± 0.001**' ± 0.072' ± 7 ± 1.2' ± 0.25' ± 0.03' ±5 ± 0.44
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Aflatoxin 0.023' 0.32 0.004' 4.7' 0.010 0.103 0.150 0.039 0.003' 0.085' 1 2.4' 0.26' 0.03' 6 0.20'
0.099 3.04 0.034 32.9 0.092 2.549 0.493 0.052 0.010 0.476 285 23.0 0.70 0.50 166 3.78
± 0.007' ± 0.32' ± 0.004' ± 4.1' ± 0.010 ± 0.605**' ± 0.101 ± 0.018 ± 0.002**' ± 0.065**' ±2 ± 1.5' ± 0.22*' ± 0.06' ± 3' ± 0.19'
Note. Single asterisk indicates intergroup difference (p < .10) at the designated timepoint. Double asterisk indicates intergroup difference (p < .05) S) at the designated timepoint. 'indicates intragroup difference (p < .05) from prephosphate loading period).
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7
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K 0) s-
-60
-40
80
100
120
Time (min) FIGURE T. Urine hydrogen ion concentration ([H + ]) during experiment 1 (i.e., after 10 d of aflatoxin treatment). Asterisk indicates significant difference (p < .05) from the control group.
dling of Ca and P, similar to those observed in the present study have been produced by decreased levels of PTH (Wideman, 1987; Glahn et al., 1988a), and hypocalcemia can be induced via vitamin D deficiency (Forte et al., 1982). The observed effects of AF on Ca and P, metabolism may reflect decreased efficacy or circulating titers of PTH (not measured) in conjunction with significant alterations in vitamin D status (Wideman, 1987). Unfortunately, an assay for avian PTH is not available (W. Burke, personal communication), and the effects of AF on PTH, Ca, and P, metabolism in a mammalian model have not been reported. Avian kidneys are both an endocrine source and a target tissue for vitamin D (Wideman, 1987). Hypocalcemia stimulates PTH secretion, which in turn stimulates renal adenylate cyclase, activating 25hydroxycholecalciferol 1a-hydroxylase to increase 1,25(OH)2D synthesis (Rasmussen et al., 1972; Fraser and Kodicek, 1973). As 1,25(OH)2D and 25(OH)D levels were decreased in the present study, it is likely that hepatic production of 25(OH)D was inhibited by AF. These observations also indicate that AF may inhibit PTH secretion and/or renal synthesis of 1,25(OH)2D. AF also may decrease the renal sensitivity to PTH and vitamin D. Interestingly, decreased renal sensitivity to PTH has been observed in vitamin D-deficient chicks (Forte et al., 1982). Vitamin D metabolites have not been shown to have direct effects on
u
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03
TABLE 4. Renal Function and Plasma Electrolyte Values (Mean ± SE) of Control and Aflatoxin-Treated Birds for Experiment 2 (i.e., 10 Days Post 10 Days of Aflatoxin or Corn Oil Treatment) 0 min after phosphate loading Values
Control
V CFR V/CFR CPAH FF Urine [ H + ] FEP FECa FENa FEK Plasma osmolality Plasma P| Total plasma Ca Blood ion Ca 2 + Plasma Na Plasma K
0.039 01.84 0.022 10.3 0.201 0.911 0.805 0.035 0.007 0.214 277 6.18 2.54 1.23 156 4.41
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.004 0.08 0.002 1.4 0.024 0.105 0.218 0.010 0.002 0.024 4 0.24 0.08 0.14 6 0.31
60 min after phosphate loading
Aflatoxin
Control
0.034 1.47 0.024 7.67 0.222 0.892 0.400 0.035 0.003 0.259 292 6.02 2.50 1.00 161 4.73
0.112 2.38 0.048 19.8 0.125 0.593 0.518 0.081 0.026 0.393 284 16.95 1.70 0.58 161 5.50
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.002 0.12** 0.002 1.3 0.037 0.159 0.176 0.008 0.001 0.048 2** 0.19 0.04 0.17 7 0.32
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.008' 0.10 0.004' 2.1' 0.011' 0.112 0.357 0.024 0.002' 0.063' 4 1.14' 0.08' 0.05' 9 1.24
120 min after phosphate loading
Aflatoxin
Control
0.089 ± 0.018*' 1.86 ± 0.40* 0.047 ± 0.003' 21.9 ± 4.7f 0.088 ± 0.010' 1.003 ± 0.218 0.541 ± 0.370 0.071 ± 0.030 0.017 ± 0.003**' 0.536 ± 0.131' 280 ± 2 15.62 ± 0.25' 2.22 ± 0.25 0.73 ± 0.07 161 ± 7 4.60 ± 0.40
0.113 2.08 0.056 24.6 0.105 0.538 0.783 0.111 0.031 0.564 291 21.10 1.24 0.43 160 3.90
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Aflatoxin 0.006' 0.19 0.006' 7.2' 0.025' 0.161 0.504 0.039 0.003' 0.074' 5' 1.73' 0.11' 0.05' 11 0.35
0.092 1.80 0.054 33.4 0.071 1.553 0.609 0.125 0.017 0.570 282 19.04 1.32 0.49 164 4.52
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.005*' 0.26 0.007' 9' 0.018' 0.528**' 0.333 0.035 0.004**' 0.104' 4' 0.92' 0.26' 0.05' 8 0.44
Note. Single asterisk indicates intergroup difference (p < .10) at the designated timepoint. Double asterisk indicates intergroup difference (p < .05) at the designated timepoint. 'indicates intragroup difference (p < .05) from prephosphate loading period).
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the renal handling of Ca in the avian kidney (Wideman, 1987); however, 1,25(OH)2D has been shown to stimulate the synthesis of a specific calcium-binding protein in avian distal tubules and collecting ducts (Narbaitz et al., 1982). Thus, 1,25(OH)2D may contribute to distal tubular finetuning of Ca excretion, and inhibition of this process by AF could account for some of the increase in FECa observed during aflatoxicosis. Aflatoxin inhibits DNA-dependent RNA polymerase and causes impairment of nucleolar DNA template function, resulting in general inhibition of protein synthesis (Gelboin et al., 1966; Yu, 1977). As a result, hypoproteinemia is a common effect of aflatoxicosis (Huff et al., 1986a, 1986b). In the present study, total plasma protein and blood ionized Ca were measured to determine if AF-induced hypoproteinemia alters blood ionized Ca, which in turn would affect circulating levels of PTH (Mueller et al., 1970). Blood ionized Ca was not altered in AF-treated animals even though total plasma protein was reduced, indicating the observed changes in the renal handling of Ca and Pi may not be due to changes in PTH synthesis induced by ionized Ca. Alternatively, AF may inhibit DNA template function in an endocrine organ such as the parathyroid gland, thereby reducing hormone synthesis and indirectly causing the observed effects on Ca and P, metabolism. In avians, phosphate accounts for approximately 25-33% of the urinary buffering capacity (Craan et al., 1982; Long and Skadhauge, 1983). At normal pH values, filtered phosphate is in the dibasic form (HPO42~). Urinary acidification titrates dibasic phosphate to its monobasic form (H2PO4~), which is excreted in the urine. In the present study, the increase in urine [H + ] during phosphate loading may reflect increased Na + /H + counterport. This effect on urine [H + ] may be secondary to an AF-induced increased in Na reabsorption, as evident by decreased FENa and increased plasma Na within the AF-treated birds of the present study (Table 3). Infusion of the 100 mM sodium phosphate provided the necessary Na and phosphate to stimulate additional Na + /H + counterport. It is interesting to note that indicators of diuresis and diarrhea, such as wet manure and increased urine flow rate, were not observed in the AFtreated birds. Therefore, increased Na reabsorption in the present study was not a secondary response to toxin-induced fluid or electrolyte loss via the kidneys or intestine. Ten days after cessation of toxin exposure, previously observed differences in V, V/GFR, FENa, FEK, and total plasma Ca were absent prior to phosphate loading (Tables 3 and 4). Also, the AF-induced effect on urine [H + ] was attenuated during the phosphate loading periods (Table 4). These results suggest that AF-related effects on the above parameters are reversible upon cessation of AF exposure. However, prior to phosphate loading GFR was decreased and plasma osmolality was increased in the AF-treated binds 10 d after AF administration was discontinued (Table 4). These differences, which were not present in experiment 1, indicate that
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AF may have prolonged effects on renal function. Decreased GFR can reflect a reduction in the functional renal mass of avian kidneys (Niznik et al., 1985; Glahn et al., 1989b). In summary, AF directly or indirectly affects Ca and P, metabolism in avians. At the present time, these effects may be related to altered vitamin D metabolism. Aflatoxicosis may also decrease endogenous PTH synthesis and decrease renal sensitivity to PTH. Alternatively, AF could extensively inhibit DNA template function such that endocrine organs such as the liver, kidney, parathyroid gland, and so forth exhibit altered sensitivity and secretion. REFERENCES Brun, C. 1957. A rapid method for determination of para-aminohippuric acid in kidney function tests. J. Lab. Clin Med. 37:955-958. Craan, A. G., Lemieux, G., Vinay, P., and Gougoux, A. 1982. The kidney of the chicken adapts. Chronic metabolic acidosis: In vivo and in vitro studies. Kidney Int. 22:103-111. Fiske, C. H., and Subbarow, Y. 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66:375-400. Forte, L. R., Langeluttig, S. G., Poelling, R. E., and Thomas, M. L. 1982. Renal parathyroid hormone receptors in the chick: Downregulation in secondary hyperparathyroid animal models. Am. J. Physiol. 242:E154-E163. Fraser, D. R., and Kodicek, E. 1973. Regulation of 25-hydroxycholecalciferol 1-hydroxylase activity in kidney by parathyroid hormone. Nature New Biol. 241:163-166. Gelboin, H. V., Wortham, J. S., Wilson, R. G., Friedman, M., and Wogan, G. N. 1966. Rapid and marked inhibition of rat-liver RNA polymerase by aflatoxin B1. Science 154:1205-1206. Glahn, R. P., Wideman, R. F., Jr. and Cowen, B. S. 1988a. Effect of gray strain infectious bronchitis virus and high dietary calcium on renal function of single comb white leghorn pullets at 6, 10, and 18 weeks of age. Poult. Sci. 67:1250-1263. Glahn, R. P., Wideman, R. F., Jr., Evangelisti, J. W., and Huff, W. E. 1988b. Effects of ochratoxin A alone and in combination with citrinin on kidney function of single comb white leghorn pullets. Poult. Sci. 67:1034-1042. Glahn, R. P., Shapiro, R. S., Vena, V. E., Wideman, R. F., Jr., and Huff, W. E. 1989a. Effects of chronic ochratoxin A and citrinin toxicosis on kidney function of single comb white leghorn pullets. Poult. Sci. 68:1205-1212. Glahn, R. P., Wideman, R. F., Jr., and Cowen, B. S. 1989b. Order of exposure to high dietary calcium and gray strain infectious bronchitis virus alters real function and the incidence of urolithiasis. Poult. Sci. 68:1193-1204. Glahn, R. P., Beers, K. W., Bottje, W. G., Wideman, R. F., Jr., and Huff, W. E. 1990. Altered renal function in broilers during aflatoxicosis. Poult. Sci. 69:1796-1797. Hnatow, L. L., and Wideman, R. F., Jr. 1985. Kidney function of single comb white leghorn pullets following acute renal portal infusion of the mycotoxin citrinin. Poult. Sci. 64:1553-1561. Hollis, B. W., and Pittard, W. B. 1984. Relative concentrations of 25-hydroxyvitamin D2/D3 dihydroxyvitamin D2/D3 in maternal plasma at delivery. Nutr. Res. 4:27. Huff, W. E., Kubena, L. F., Harvey, R. B., Hagler, W. M., Jr., Swanson, S. P., Phillips, T. D., and Creger, C. R. 1986a. Individual and combined effects of aflatoxin and deoxynivalenol (DON) in broiler chickens. Poult. Sci. 65:1291-1298. Huff, W. E., Kubena, L. F., Harvey, R. B., Corrier, D. E., and Mollenhauer, H. H. 1986b. Progression of aflatoxicosis in broiler chickens. Poult. Sci. 65:1891-1899. Huff, W. E., and Doerr, J. A. 1981. Synergism between aflatoxin and ochratoxin A in broiler chickens. Poult. Sci. 60:550-555.
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