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Soy-deficient diet induces renal lesions in juvenile rats
Food and Chemical Toxicology 121 (2018) 467–471
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Soy-deficient diet induces renal lesions in juvenile rats a
b
a,∗
c
Eli Parker , Pallavi McElroy , Catherine Picut , Kathleen Szabo , Stephanie White-Hunt a b c
T a
Charles River Laboratories, Inc, 4025 Stirrup Creek Drive, Suite 150, Durham, NC, 27703, USA Charles River Laboratories, Inc, 1407 George Road, Ashland, OH, 44805, USA Charles River Laboratories Inc, 15 Worman's Mill Court, Suite I, Frederick, MD, 21701, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Juvenile Isoflavone Preclinical Toxicity Diet Mineralization Basophilic tubules
Certified LabDiet® 5K96 Advanced Protocol™ Verified Casein Diet 10 IF (5K96) is a commercial diet low in soy isoflavones developed for use in developmental and reproductive toxicity (DART) studies, especially those designed to detect endocrine disruptors. The objective of this study was to determine the incidences and severities of 5K96-associated renal lesions in control F0 and F1 cohorts of rats fed the 5K96 diet. Kidneys from control animals of four DART studies involving Sprague-Dawley rats fed the 5K96 diet, were evaluated microscopically. Mineralization and basophilic tubules were present in high incidence/severity in males and females compared to historical controls fed conventional diets. F1 cohorts were affected to a far greater degree than F0 cohorts, and females were affected more than males. Consideration of target tissue and mode of action should be given before automatically incorporating the 5K96 diet into DART study designs, and caution should be exercised when identifying and interpreting renal toxicity in the F1 cohorts of such studies.
1. Introduction Diet has been known to both exacerbate and mitigate spontaneous kidney disease of adult rats. Increased incidence and severity of chronic progressive nephropathy (CPN), a background tubulo-interstitial lesion primarily in adult male rats, has been attributed to high protein and/or caloric content of certain diets (Hard et al., 2013; Keenan et al., 2000). On the other hand, decreased severity of CPN has been associated with diets containing soy as its protein source, since soy contains isoflavones with estrogenic-like activity (Rao, 2002). Since males are primarily affected with CPN and soy mitigates CPN, it is apparent female hormones protect against certain tubulo-interstitial diseases of the kidney. In order to minimize the occurrence of CPN in adult rats on preclinical safety studies, most conventional rodent diets (e.g., LabDiet® 5001) are soy-based with controlled protein and caloric content. Mineralization is another background lesion in rats that can be exacerbated or mitigated by diet. Diets with a calcium: phosphorus (Ca:P) molar ratio < 1.00 have been associated with mineralization, especially in adult female rats (Reeves et al., 1993). Unlike the situation with CPN where female hormones mitigate disease, female hormones promote mineralization (Rao, 2002; Reeves et al., 1993). In order to minimize the occurrence of mineralization in adult female rats on
preclinical safety studies, commercial rodent diets with a Ca:P molar ratio of at least 1.3 are commonly used (Reeves, 1997; Reeves et al., 1993). Even though most commercial diets have been successfully adjusted to control protein and caloric intake (to help minimize CPN), and to optimize Ca:P ratio (to help minimize mineralization) in the adult rat, these adjustments may not sufficiently protect the developing juvenile kidney from renal lesions. In a series of extended one-generation reproductive toxicity (EOGRT) studies at Charles River, we recognized that a soy-free commercial diet was associated with exceptionally high incidences and severities of basophilic tubules and mineralization in the control F1 cohorts, which were fed 5K96 from the time of weaning, as compared to the F0 adult cohorts. Regardless of cohort, females were affected to a greater degree than males for both types of lesions. These observations prompted a more complete retrospective analysis of rat kidneys from four DART studies, each of which employed the same soyfree diet. The purposes of this analysis was to: i) determine the incidence and severity of renal lesions in various cohorts of rats on DART studies employing soy-free diets; ii) provide historical control data for future studies utilizing this diet; and iii) highlight how 5K96-induced renal lesions may complicate interpretation of test article-related renal toxicity.
Abbreviations: BUN, Blood Urea Nitrogen; CPN, Chronic Progressive Nephropathy; Cr, Creatinine; DART, Developmental And Reproductive Toxicity; EOGRT, Extended One-Generation Reproductive Toxicity; H&E, Hematoxylin and Eosin; GLP, Good Laboratory Practices; OECD, Office of Economic Cooperation and Development; PND, postnatal day; U.S. FDA, United States Food and Drug Administration ∗ Corresponding author. Charles River Laboratories, Suite 150, 4025 Stirrup Creek Drive, Durham, NC, 27703, USA. E-mail address: [email protected] (C. Picut). https://doi.org/10.1016/j.fct.2018.09.044 Received 16 August 2018; Received in revised form 18 September 2018; Accepted 20 September 2018 Available online 21 September 2018 0278-6915/ © 2018 Elsevier Ltd. All rights reserved.
Food and Chemical Toxicology 121 (2018) 467–471
E. Parker et al.
Fig. 1. 5K96-Induced Renal Mineralization. This series of images depict renal mineralization (moderate) at the cortico-medullary junction in an F1 control female fed 5K96 diet. (A) There was a dark blue band of mineral between the cortex and the outer stripe of the medulla. 1.0 x, H&E. (B) There was no disruption of the adjacent parenchyma. 8.0 x, H&E. (C) This mineral was strongly positive for calcium. 6.0 x, Alizarin Red. (D) The mineral was moderately positive for phosphate. 9.5 x, von Kossa. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
the severities of findings were based on a 5 - point grading scale (1: minimal, 2: mild, 3: moderate, 4: marked, 5: severe). The average (mean) incidence and average severity of each finding in each study was determined. Data from the control groups from each of the four DART studies were compared to Charles River-Ashland historical control data set for Crl:CD(SD) rats (updated August–September 2015). This historical control data set is based on studies conducted at Charles River-Ashland in Sprague Dawley rats fed commercially available conventional rodent diet, most commonly but not exclusively, Certified Rodent LabDiet® 5001 (Purina Mills International Nutrition, St. Louis, MO). The studies were conducted at Charles River - Ashland in compliance with OECD Principles of Good Laboratory Practice (GLP) (Office of Economic Cooperation and Development (OECD), 1998), and U.S. Code of Federal Regulations (21 CFR Part 58; 50 CFR Parts 160 and 792). The use of animals was carried out in accordance with Guide for the Care and Use of Laboratory Animals (National Research Council (NRC), 2011).
2. Materials and methods Four DART studies, each involving Sprague-Dawley rats [(Cr1:CD (SD) rats from Charles River Laboratories)] fed the soy free diet Certified Rodent LabDiet® 5K96 Advanced Protocol™ Verified Casein Diet 10 1F (5K96; PMI Nutrition, St. Louis, MO) were reviewed. Each study was designed in compliance with Office of Economic Cooperation and Development (OECD) Guidelines 443: Extended One-Generation Reproductive Toxicity (EOGRT) studies (Office of Economic Cooperation and Development (OECD), 1998). F0 cohorts (adult males and females) were fed the 5K96 diet ad libitum during acclimation, mating, pregnancy, and lactation (for a minimum of 10 weeks), and F1 cohorts (i.e., rat pups) were fed this same 5K96 diet for approximately 10 weeks, from weaning (PND 21–22) until scheduled necropsy (approximately PND 90 or 12 weeks of age). Each control and test article-treated group on these studies had 25 animals of each sex. The route of administration of test article varied between studies. Test article was administered by whole body inhalation (Study 1), by oral dietary administration (Study 2), or by oral gavage (Studies 3 and 4). For each of the test articles in Studies 1 through 4, the kidneys or any portion of the urinary system were not considered a potential target tissue. In the interest of confidentiality, test article identity cannot be revealed in this report, but since this report is limited to the evaluation of control animals, test article identity is not relevant. Complete hematology, serum chemistry and urinalysis parameters were obtained prior to necropsy following overnight fasting, and kidney weights (along with body weights and multiple organ weights) were obtained at necropsy. Five μm sections of both kidneys from control and test articletreated F0 and F1 males and females were fixed in 10% formalin, processed routinely, and stained with hematoxylin and eosin (H&E). Selected tissues were stained with von Kossa and/or alizarin red to help characterize the nature of the mineral deposits. The slides were evaluated microscopically by board-certified veterinary pathologists, and
3. Results In the control F0 and F1 females and in F1 males, 5K96 diet was associated with increased incidence and/or severity of renal mineralization and basophilic tubules, when compared to age- and sex-matched historical controls (Figs. 1 and 2; Table 1). Females were affected to a greater extent than males for both lesions, and F1 cohorts were affected to a greater extent than F0 cohorts. Mineralization was characterized by deposition of mineral at the corticomedullary junction, along the outer most portion of the outer stripe of the medulla (Fig. 1). Small deposits of mineralization were occasionally present in the inner stripe of the medulla. At all severities present (minimal to marked), the mineral did not cause degenerative changes in adjacent parenchyma. Mineral stained strongly positive with alizarin red (which stains calcium) (Puchtler et al., 1969) and moderately strongly positive with von Kossa (which is nonspecific for calcium 468
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exacerbation (increased incidence and severity with increased dose) of basophilic tubules in F1 females in two studies (Studies 1 and 2), and this exacerbation corresponded to higher group mean kidney weights in Study 2 (data not shown). In Study 2, there was also such a dose response profile of increased incidence and severity of mineralization in both F1 males and females (data not shown). Other than incidence and severity, the renal lesions in the treated groups were similar in morphologic appearance to that in the control groups. 4. Discussion Certified LabDiet® 5K96 Advanced Protocol™ Verified Casein Diet 10 IF (5K96) is a commercial diet low in soy isoflavones developed for use in developmental and reproductive toxicity (DART) studies, especially those designed to detect endocrine disruptors (Thigpen et al., 2004). In comparing the composition of 5K96 to conventional LabDiet® 5001 (Table 3), 5K96 is lower in protein, higher in calcium and phosphorus, has a lower Ca:P ratio, and is free of soy isoflavones. Casein, the protein source for 5K96, is naturally high in phosphorus; and additional calcium is added to the 5K96 diet to keep an optimal Ca: P ratio approximating 1.3 (Reeves et al., 1993). Therefore, 5K96 is a diet relatively high in both Ca and P, yet has a relatively low Ca:P ratio. A high incidence of mineralization has been reported in other published studied utilizing 5K96 diet, though the focus of these other publications were to report estrogen-related effects of test article, and not the occurrence, incidence or severity of diet-induced lesions. In reviewing the data in those publications, renal mineralization was present in 80% of F0 and 84% of F1 control females (National Institutes of Health, 2010); in 86% of F1 control female rats at PND 50 (National Institutes of Health, 2007); and in 73–100% of treated and control female rats at PND 50 (Latendresse et al., 2001). Despite these reportedly high incidences of mineralization, no association between 5K96 and mineralization was acknowledged by the authors. The 5K96-induced mineralization, reported herein, is similar morphologically to the mineralization reported as a spontaneous occurrence in adult female rats, as both occur at the corticomedullary junction. One difference between the 5K96-induced mineral and that which occurs spontaneously is that the spontaneous lesion (in rats fed conventional soy-based diets) is of low incidence (< 20% in F1 females) and minimal severity. The pathogenesis of the 5K96-induced mineralization likely reflects something other than a mere Ca:P ratio imbalance. After all, the Ca:P ratio of 5K96 is only slightly less than 1.3, the level reported by Reeves et al. (1993) to prevent nephrocalcinosis. It is possible that any diet-induced changes in fluid balance in control animals, especially in the weanlings at PND 21 with immature kidney function, could precipitate mineralization. In fact, soy-free diets have been associated with altered fluid balance by causing increased urine output and decreased fluid resorption by the tubules (Cooper et al., 2006). It is also possible that exposure to 5K96 before the loop of Henle is mature may represent a vulnerable time for the kidney in terms of mineral balance. In the thick ascending limb of the loop of Henle, claudins control the passive paracellular transport of calcium and magnesium, and a deficiency in claudins cause nephrocalcinosis of the outer and inner stripes of the medulla and hypermagnesemia (Breiderhoff et al., 2012). Others have reported the involvement of magnesium balance with nephrocalcinosis (Reeves et al., 1993). Since the loop of Henle does not morphologically mature until PND 28 (Frazier, 2017; Brown et al., 2016), the first week of feeding 5K96 to F1 cohorts (PND 21 – PND 28) may render that animal susceptible to nephrocalcinosis. The basophilic tubules associated with 5K96, reported herein, can appear microscopically similar to, and be easily confused with, chronic progressive nephropathy (CPN), an exceedingly common finding in rats. After all, both are characterized by basophilic tubules. But in CPN, prominent features also include peritubular basement membranes, tubular dilatation, intraluminal protein, hyaline casts, and
Fig. 2. 5K96-Induced Basophilic Tubules. Basophilic tubules (moderate) in the kidney of F1 control female rat fed 5K96 diet. (A) The kidneys were characterized by multifocal collections of basophilic tubules limited to the cortex. These tubules were occasionally dilated (arrowhead). 15 x, H&E. (B) The basophilic tubules had crowded nuclei with slight thickening of basement membranes (arrows). The tubular lumina occasionally had pink intracellular proteinaceous fluid. There is minimal mononuclear cell infiltration into the interstitium associated with affected tubules. 33 x, H&E. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
and more selective for phosphate) (Meloan and Puchtler, 1985; Rungby et al., 1993). Mineralization was consistently at an incidence of 100% in the F1 females, and commonly graded as moderate to marked, with a group mean severity as high as 3.3. The term “basophilic tubules” was a morphologic diagnosis that was characterized by multifocal discrete clusters of tinctorially abnormal tubules in the outer cortex (Fig. 2). The main feature of this lesion was multifocal clusters of tubules lined by epithelial cells having deeply basophilic cytoplasm and crowded nuclei. Other less common features included prominent tubular basement membranes and dilated tubular lumina. Rarely, there was intraluminal protein and interstitial mononuclear inflammatory cell infiltration. The outer cortical location and appearance of the affected tubules were most consistent with that of distal convoluted tubules. The proximal convoluted tubules in the cortex and those tubules within the medulla (loops of Henle and collecting ducts) appeared normal microscopically. The segments of loops of Henle at the corticomedullary junction were commonly effaced by mineral. The incidence of basophilic tubules ranged from 85 to 93% in F1 females, commonly at moderate severity, and the average group severity was up to 1.85. In general, the degree of basophilic tubules paralleled the degree of mineralization, but this correlation was not invariable. The mean kidney weights, blood urea nitrogen (BUN) and serum creatinine values for the four DART studies are provided in Table 2. In all studies, these values were within the Charles River-Ashland historical control range of the study means. While the focus of this publication is to report renal lesions in control animals fed 5K96, these diet-induced lesions were exacerbated in the treated groups of animals. In particular there was test article-related 469
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Table 1 Incidence and severity of renal Lesionsa in control F0 and F1 rats from four DART studies.
Table 2 Mean kidney weights, urea nitrogen, and serum creatinine values in control F0 and F1 rats from four DART studies. F0 Female
Study 1a Study 2 Study 3 Study 4 Historical Control1
Study 1 Study 2 Study 3 Study 4 HistoricalControl1
F0 Males
Age
Kidney Weight (g)
Urea Nitrogen (mg/ dL)
Creatinine (mg/dL)
Kidney Weight (g)
Urea Nitrogen (mg/ dL)
Creatinine (mg/dL)
19–21 wks 19–21 wks 19–21 wks 19–21 wks 19–21 wks
1.8 2.0 2.0 2.2 2.1 (1.6–2.5)
14.4 16.3 18.2 15.4 15.5 (12.2–20.2)
0.4 0.4 0.5 0.5 0.4 (0.4–0.5)
3.0 3.6 3.7 3.8 3.7 (2.8–4.6)
11.7 13.1 14.8 12.0 14.5 (9.0–17.5)
0.4 0.4 0.4 0.4 0.3 (0.3–0.5)
Age
F1 Female Kidney Weight (g)
Urea Nitrogen (mg/ dL)
Creatinine Values (mg/ dL)
F1 Males Kidney Weight (g)
Urea Nitrogen (mg/ dL)
Creatinine Values (mg/ dL)
12 wks 12 wks 12 wks 12 wks 9–12 wks
1.7 2.0 2.0 2.0 1.9 (1.4–2.3)
14.3 14.4 17.1 14.2 15.3 (11.0–20.3)
0.4 0.4 0.4 0.4 0.3 (0.2–0.5)
2.6 3.3 3.4 3.3 2.9 (2.0–3.9)
12.5 11.8 14.0 13.7 13.3 (9.4–17.2)
0.3 0.3 0.4 0.4 0.3 (0.2–0.4)
1
Group mean values for historical control is provided. The numbers in the parentheses refer to either the range of 2 standard deviations from the mean (kidney weights) or range of the study means (urea nitrogen and creatinine). a The control group size for each study was 25 animals per group per sex.
pathophysiologic differences between these lesions. CPN is a lesion of older male rats, while the 5K96-induced basophilic tubules are more prominent in young females (< 12 weeks of age). While both CPN and basophilic tubules are mitigated with a soy-based diet, the protective
inflammatory cell infiltration (Hard et al., 2013). These additional features were minor and variable in the 5K96-induced basophilic tubules reported herein. Not only are there distinct morphologic features between CPN and 5K96-induced basophilic tubules, there are distinct 470
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studies. If 5K96 diet is included in the study design, special consideration may be needed to interpret any dose response increase in renal lesions.
Table 3 Composition of soy-free 5K96 diet and conventional 5001 rodent diets.
Protein source Protein (%) Ca (%) P (%) P -non-phytate (%) Ca:P b Magnesium (%)
LabDiet® 5K96
LabDiet® 5001
Soy-Free
Soy – Containing
Casein 19.1 1.05 0.93 0.71 1.29 0.20
Soybean meal 24.1 0.95 0.68 0.42 1.39 0.21
a
Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.fct.2018.09.044. References Breiderhoff, T., Himmerkus, N., Stuiver, M., Mutig, K., Will, C., Meij, I.C., Bachmann, S., Bleich, M., Willnow, T.E., Muller, D., 2012. Deletion of claudin-10 (Cldn10) in the thick ascending limb impairs paracellular sodium permeability and leads to hypermagnesemia and nephrocalcinosis. Proc. Natl. Acad. Sci. Unit. States Am. 109, 14241–14246. Brown, D.L., Walling, B.E., Mattix, M.E., 2016. The urinary system. In: Atlas of Histology of the Juvenile Rat (Parker GA and Picut CA. Elsevier, San Diego, CA, pp. 395–421 (Chapter 13). Camacho, L., Lewis, S.M., Vanlandingham, M.M., Juliar, B.E., et al., 2017. Comparison of endpoints relevant to toxicity assessment in 3 generations of CD1- mice. Food Chem. Toxicol. 94, 39–56. Cooper, S., Latendresse, J.R., Doerge, D.R., Twaddle, N.C., Fu, X., Delclos, K.B., 2006. Dietary modulation of p-nonylphenol-induced polycystic kidneys in male spraguedawley rats. Toxicol. Sci. 91, 631–642. Frazier, K.S., 2017. Review article. Species differences in renal development and associated developmental nephrotoxicity. Birth Defects Res Part B 109, 1243–1256. Hard, G.C., Banton, M.I., Bretzlaff, R.S., et al., 2013. Consideration of rat chronic progressive nephropathy in regulatory evaluations for carcinogenicity. Toxicol. Sci. 132, 268–275. Keenan, K.P., Coleman, J.B., McCoy, C.L., Hoe, C.M., Soper, K.A., Laroque, P., 2000. Chronic nephropathy in ad libitum overfed Sprague Dawley rats and its early attenuation by increasing degrees of dietary (caloric) restriction to control growth. Toxicol. Pathol. 28, 788–798. Latendresse, J.R., Newbold, R.R., Weis, C.C., Delclos, K.B., 2001. Polycystic Kidney Disease Induced in F1 Sprague-Dawley Rats Fed para-Nonylphenol in a Soy-Free, Casein-Containing Diet. Toxicol. Sci. 62, 140–147. Meloan, S.N., Puchtler, H., 1985. Chemical mechanisms of staining methods: von Kossa's technique. What von Kossa really wrote and a modified reaction for selective demonstration of inorganic phosphate. J. Histotechnol. 8, 11–13. Moore, R.J., Reeves, P.G., Veum, T.L., 1984. Influence of dietary phosphorus and sulphaguanidine levels on P utilization in rats. Br. J. Nutr. 51, 453–465. National Institutes of Health, 2007. NTP Technical Report on the Reproductive Dose Range-finding Toxicity Study of Genistein (CAS No. 446-72-0) Administered in Feed to Sprague-Dawley Rats. Public Health Service, U.S. Department of Health and Human Services Toxicity Report Series Number 79. National Institutes of Health, 2010. NTP Technical Report on the Toxicology and Carcinogenesis Study of Ethinyl Estradiol (CAS No. 57-63-6) in Sprague-Dawley Rats. Public Health Service, U.S. Department of Health and Human Services, NIH Publication No, pp. 10–5889. National Research Council (NRC), 2011. Guide for the Care and Use of Laboratory Animals, Eight Edition. National Academies Press, Washington, D.C. https://doi.org/ 10.17226/12910. Office of Economic Cooperation and Development (OECD), 1998. Principles of Good Laboratory Practice (GLP) ENV/MC/CHEM (98)17. OECD Publishing, Paris revised in 1997. Philbrick, D.J., Bureau, D.P., Collins, F.W., Holub, B.J., 2003. Evidence that soyasaponin Bb retards disease progression in a murine model of polycystic kidney disease. Kidney Int. 63, 1230–1239. Puchtler, H., Meloan, S.N., Terry, M.S., 1969. On the history and mechanism of alizarin and alizarin Red S Stains for Calcium. J. Histochem. Cytochem. 17, 110–124. Ranich, T., Bhathena, S.J., Velasquez, M.T., 2001. Protective effects of dietary phytoestrogens in chronic renal disease. J. Ren. Nutr. 11, 183–193. Rao, G.N., 2002. Diet and kidney diseases in rats. Toxicol. Pathol. 30, 651–656. Reeves, P.G., 1997. Components of the AIN-93 diets as improvements in the AIN-76A diet. J. Nutr. 127, 838S–841S. Reeves, P.G., Rossow, K.L., Lindlauf, J., et al., 1993. Development and testing of the AIN93 purified diets for rodents: results on growth, kidney calcification and bone mineralization in rats and mice. J. Nutr. 123, 1923–1931. Rungby, J., Kassem, M., Eriksen, E.F., Danscher, G., 1993. The von Kossa reaction for calcium deposits: sliver lactate staining increases sensitivity and reduces background. Histochem. J. 25, 446–451. Thigpen, J.E., Setchell, K.D., Saunders, H.E., Haseman, J.K., Gran, M.G., Forsythe, D.B., 2004. Selecting the appropriate rodent diet for endocrine disruptor research and testing studies. ILAR J. 45, 401–416. Travlos, G.S., Hard, G.C., Betz, L.J., Kissling, G.E., 2011. Chronic progressive nephropathy in male F344 rats in 90-day toxicity studies: its occurrence and association with renal tubule tumors in subsequent 2-year bioassays. Toxicol. Pathol. 38, 381–389.
a
Contains isoflavones genistein (86.90 ppm), daidzein (50.47 ppm), and coumestral (6.88 ppm) (CamachoLewis et al., 2017) b Calculated as Calcium:total Phosphorus. However, in monogastric animals, phytate phosphorus is less “available” following digestion than non-phytate phosphorus. The higher the non-phytate phosphorus, the more P that is available, thus lowering the effective Ca:P ratio (Moore et al., 1984; Reeves et al., 1993).
mechanism is likely different. With CPN, the protective effect is linked to the estrogenic isoflavones of soy (Ranich et al., 2001; Philbrick et al., 2003). But since 5K96-induced basophilic tubules are seen primarily in females (with natural circulating levels of estrogen), the protective factor in soy is apparently not the estrogenic isoflavones. It is not clear if 5K96-induced basophilic tubules represent a degenerative or a metabolic change (as is CPN), delayed development (since most tubules in the early postnatal rat up to PND 14 are basophilic) (Brown et al., 2016), or a retrograde nephropathy in response perhaps to mineral imbalance in the thick ascending limb of the loop of Henle. What is clear is there is active morphologic and functional renal development ongoing at the time of early exposure of F1 cohorts to 5K96 (PND 21 – PND 90). Renal morphologic maturation in rats is not complete until PND 28 and functional maturation is not complete until PND 42 (acid base equilibrium is achieved by PND 27; organic anion transporters are mature by PND 35; and stable GFR is achieved by PND 35–42) (Frazier, 2017). These ongoing developmental changes could render the kidney unusually susceptible to certain diets. Regardless of the pathogenesis, these basophilic tubules can mimic toxicity, or they can confound the interpretation or severity of co-existent toxicity. The dose response profiles for basophilic tubules and mineralization, primarily in the F1 female cohorts in two of the four EOGRT studies, do not necessarily suggest direct test article-related toxicity. Given the fact that soy free diets may alter fluid homeostasis, and are associated with a high background incidence of mineralization and basophilic tubules in control F1 animals, these animals are not in homeostasis. It is foreseeable that any additional effect of test article, albeit minor and nonspecific, could exacerbate any pre-existing diet-induced renal lesions, in the same way that some test articles exacerbate CPN (Travlos et al., 2011). 5. Conclusion There is high incidence and severity of basophilic tubules and renal mineralization in studies employing a soy-free 5K96 diet, especially in F1 females fed from weaning up to PND 90. An exaggerated reaction to 5K96 diet in F1 females, when compared to F0 females, for both mineralization and basophilic tubules likely reflects the fact that F1 animals are being exposed to 5K96 diet during morphologic and functional postnatal renal development. Since 5K96-associated renal lesions may complicate interpretation of test article-related renal effects, especially in young F1 or juvenile animals, caution should be exercised before incorporating the 5K96 diet into preclinical DART or juvenile safety
471
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Soy-deficient diet induces renal lesions in juvenile rats a
b
a,∗
c
Eli Parker , Pallavi McElroy , Catherine Picut , Kathleen Szabo , Stephanie White-Hunt a b c
T a
Charles River Laboratories, Inc, 4025 Stirrup Creek Drive, Suite 150, Durham, NC, 27703, USA Charles River Laboratories, Inc, 1407 George Road, Ashland, OH, 44805, USA Charles River Laboratories Inc, 15 Worman's Mill Court, Suite I, Frederick, MD, 21701, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Juvenile Isoflavone Preclinical Toxicity Diet Mineralization Basophilic tubules
Certified LabDiet® 5K96 Advanced Protocol™ Verified Casein Diet 10 IF (5K96) is a commercial diet low in soy isoflavones developed for use in developmental and reproductive toxicity (DART) studies, especially those designed to detect endocrine disruptors. The objective of this study was to determine the incidences and severities of 5K96-associated renal lesions in control F0 and F1 cohorts of rats fed the 5K96 diet. Kidneys from control animals of four DART studies involving Sprague-Dawley rats fed the 5K96 diet, were evaluated microscopically. Mineralization and basophilic tubules were present in high incidence/severity in males and females compared to historical controls fed conventional diets. F1 cohorts were affected to a far greater degree than F0 cohorts, and females were affected more than males. Consideration of target tissue and mode of action should be given before automatically incorporating the 5K96 diet into DART study designs, and caution should be exercised when identifying and interpreting renal toxicity in the F1 cohorts of such studies.
1. Introduction Diet has been known to both exacerbate and mitigate spontaneous kidney disease of adult rats. Increased incidence and severity of chronic progressive nephropathy (CPN), a background tubulo-interstitial lesion primarily in adult male rats, has been attributed to high protein and/or caloric content of certain diets (Hard et al., 2013; Keenan et al., 2000). On the other hand, decreased severity of CPN has been associated with diets containing soy as its protein source, since soy contains isoflavones with estrogenic-like activity (Rao, 2002). Since males are primarily affected with CPN and soy mitigates CPN, it is apparent female hormones protect against certain tubulo-interstitial diseases of the kidney. In order to minimize the occurrence of CPN in adult rats on preclinical safety studies, most conventional rodent diets (e.g., LabDiet® 5001) are soy-based with controlled protein and caloric content. Mineralization is another background lesion in rats that can be exacerbated or mitigated by diet. Diets with a calcium: phosphorus (Ca:P) molar ratio < 1.00 have been associated with mineralization, especially in adult female rats (Reeves et al., 1993). Unlike the situation with CPN where female hormones mitigate disease, female hormones promote mineralization (Rao, 2002; Reeves et al., 1993). In order to minimize the occurrence of mineralization in adult female rats on
preclinical safety studies, commercial rodent diets with a Ca:P molar ratio of at least 1.3 are commonly used (Reeves, 1997; Reeves et al., 1993). Even though most commercial diets have been successfully adjusted to control protein and caloric intake (to help minimize CPN), and to optimize Ca:P ratio (to help minimize mineralization) in the adult rat, these adjustments may not sufficiently protect the developing juvenile kidney from renal lesions. In a series of extended one-generation reproductive toxicity (EOGRT) studies at Charles River, we recognized that a soy-free commercial diet was associated with exceptionally high incidences and severities of basophilic tubules and mineralization in the control F1 cohorts, which were fed 5K96 from the time of weaning, as compared to the F0 adult cohorts. Regardless of cohort, females were affected to a greater degree than males for both types of lesions. These observations prompted a more complete retrospective analysis of rat kidneys from four DART studies, each of which employed the same soyfree diet. The purposes of this analysis was to: i) determine the incidence and severity of renal lesions in various cohorts of rats on DART studies employing soy-free diets; ii) provide historical control data for future studies utilizing this diet; and iii) highlight how 5K96-induced renal lesions may complicate interpretation of test article-related renal toxicity.
Abbreviations: BUN, Blood Urea Nitrogen; CPN, Chronic Progressive Nephropathy; Cr, Creatinine; DART, Developmental And Reproductive Toxicity; EOGRT, Extended One-Generation Reproductive Toxicity; H&E, Hematoxylin and Eosin; GLP, Good Laboratory Practices; OECD, Office of Economic Cooperation and Development; PND, postnatal day; U.S. FDA, United States Food and Drug Administration ∗ Corresponding author. Charles River Laboratories, Suite 150, 4025 Stirrup Creek Drive, Durham, NC, 27703, USA. E-mail address: [email protected] (C. Picut). https://doi.org/10.1016/j.fct.2018.09.044 Received 16 August 2018; Received in revised form 18 September 2018; Accepted 20 September 2018 Available online 21 September 2018 0278-6915/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. 5K96-Induced Renal Mineralization. This series of images depict renal mineralization (moderate) at the cortico-medullary junction in an F1 control female fed 5K96 diet. (A) There was a dark blue band of mineral between the cortex and the outer stripe of the medulla. 1.0 x, H&E. (B) There was no disruption of the adjacent parenchyma. 8.0 x, H&E. (C) This mineral was strongly positive for calcium. 6.0 x, Alizarin Red. (D) The mineral was moderately positive for phosphate. 9.5 x, von Kossa. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
the severities of findings were based on a 5 - point grading scale (1: minimal, 2: mild, 3: moderate, 4: marked, 5: severe). The average (mean) incidence and average severity of each finding in each study was determined. Data from the control groups from each of the four DART studies were compared to Charles River-Ashland historical control data set for Crl:CD(SD) rats (updated August–September 2015). This historical control data set is based on studies conducted at Charles River-Ashland in Sprague Dawley rats fed commercially available conventional rodent diet, most commonly but not exclusively, Certified Rodent LabDiet® 5001 (Purina Mills International Nutrition, St. Louis, MO). The studies were conducted at Charles River - Ashland in compliance with OECD Principles of Good Laboratory Practice (GLP) (Office of Economic Cooperation and Development (OECD), 1998), and U.S. Code of Federal Regulations (21 CFR Part 58; 50 CFR Parts 160 and 792). The use of animals was carried out in accordance with Guide for the Care and Use of Laboratory Animals (National Research Council (NRC), 2011).
2. Materials and methods Four DART studies, each involving Sprague-Dawley rats [(Cr1:CD (SD) rats from Charles River Laboratories)] fed the soy free diet Certified Rodent LabDiet® 5K96 Advanced Protocol™ Verified Casein Diet 10 1F (5K96; PMI Nutrition, St. Louis, MO) were reviewed. Each study was designed in compliance with Office of Economic Cooperation and Development (OECD) Guidelines 443: Extended One-Generation Reproductive Toxicity (EOGRT) studies (Office of Economic Cooperation and Development (OECD), 1998). F0 cohorts (adult males and females) were fed the 5K96 diet ad libitum during acclimation, mating, pregnancy, and lactation (for a minimum of 10 weeks), and F1 cohorts (i.e., rat pups) were fed this same 5K96 diet for approximately 10 weeks, from weaning (PND 21–22) until scheduled necropsy (approximately PND 90 or 12 weeks of age). Each control and test article-treated group on these studies had 25 animals of each sex. The route of administration of test article varied between studies. Test article was administered by whole body inhalation (Study 1), by oral dietary administration (Study 2), or by oral gavage (Studies 3 and 4). For each of the test articles in Studies 1 through 4, the kidneys or any portion of the urinary system were not considered a potential target tissue. In the interest of confidentiality, test article identity cannot be revealed in this report, but since this report is limited to the evaluation of control animals, test article identity is not relevant. Complete hematology, serum chemistry and urinalysis parameters were obtained prior to necropsy following overnight fasting, and kidney weights (along with body weights and multiple organ weights) were obtained at necropsy. Five μm sections of both kidneys from control and test articletreated F0 and F1 males and females were fixed in 10% formalin, processed routinely, and stained with hematoxylin and eosin (H&E). Selected tissues were stained with von Kossa and/or alizarin red to help characterize the nature of the mineral deposits. The slides were evaluated microscopically by board-certified veterinary pathologists, and
3. Results In the control F0 and F1 females and in F1 males, 5K96 diet was associated with increased incidence and/or severity of renal mineralization and basophilic tubules, when compared to age- and sex-matched historical controls (Figs. 1 and 2; Table 1). Females were affected to a greater extent than males for both lesions, and F1 cohorts were affected to a greater extent than F0 cohorts. Mineralization was characterized by deposition of mineral at the corticomedullary junction, along the outer most portion of the outer stripe of the medulla (Fig. 1). Small deposits of mineralization were occasionally present in the inner stripe of the medulla. At all severities present (minimal to marked), the mineral did not cause degenerative changes in adjacent parenchyma. Mineral stained strongly positive with alizarin red (which stains calcium) (Puchtler et al., 1969) and moderately strongly positive with von Kossa (which is nonspecific for calcium 468
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exacerbation (increased incidence and severity with increased dose) of basophilic tubules in F1 females in two studies (Studies 1 and 2), and this exacerbation corresponded to higher group mean kidney weights in Study 2 (data not shown). In Study 2, there was also such a dose response profile of increased incidence and severity of mineralization in both F1 males and females (data not shown). Other than incidence and severity, the renal lesions in the treated groups were similar in morphologic appearance to that in the control groups. 4. Discussion Certified LabDiet® 5K96 Advanced Protocol™ Verified Casein Diet 10 IF (5K96) is a commercial diet low in soy isoflavones developed for use in developmental and reproductive toxicity (DART) studies, especially those designed to detect endocrine disruptors (Thigpen et al., 2004). In comparing the composition of 5K96 to conventional LabDiet® 5001 (Table 3), 5K96 is lower in protein, higher in calcium and phosphorus, has a lower Ca:P ratio, and is free of soy isoflavones. Casein, the protein source for 5K96, is naturally high in phosphorus; and additional calcium is added to the 5K96 diet to keep an optimal Ca: P ratio approximating 1.3 (Reeves et al., 1993). Therefore, 5K96 is a diet relatively high in both Ca and P, yet has a relatively low Ca:P ratio. A high incidence of mineralization has been reported in other published studied utilizing 5K96 diet, though the focus of these other publications were to report estrogen-related effects of test article, and not the occurrence, incidence or severity of diet-induced lesions. In reviewing the data in those publications, renal mineralization was present in 80% of F0 and 84% of F1 control females (National Institutes of Health, 2010); in 86% of F1 control female rats at PND 50 (National Institutes of Health, 2007); and in 73–100% of treated and control female rats at PND 50 (Latendresse et al., 2001). Despite these reportedly high incidences of mineralization, no association between 5K96 and mineralization was acknowledged by the authors. The 5K96-induced mineralization, reported herein, is similar morphologically to the mineralization reported as a spontaneous occurrence in adult female rats, as both occur at the corticomedullary junction. One difference between the 5K96-induced mineral and that which occurs spontaneously is that the spontaneous lesion (in rats fed conventional soy-based diets) is of low incidence (< 20% in F1 females) and minimal severity. The pathogenesis of the 5K96-induced mineralization likely reflects something other than a mere Ca:P ratio imbalance. After all, the Ca:P ratio of 5K96 is only slightly less than 1.3, the level reported by Reeves et al. (1993) to prevent nephrocalcinosis. It is possible that any diet-induced changes in fluid balance in control animals, especially in the weanlings at PND 21 with immature kidney function, could precipitate mineralization. In fact, soy-free diets have been associated with altered fluid balance by causing increased urine output and decreased fluid resorption by the tubules (Cooper et al., 2006). It is also possible that exposure to 5K96 before the loop of Henle is mature may represent a vulnerable time for the kidney in terms of mineral balance. In the thick ascending limb of the loop of Henle, claudins control the passive paracellular transport of calcium and magnesium, and a deficiency in claudins cause nephrocalcinosis of the outer and inner stripes of the medulla and hypermagnesemia (Breiderhoff et al., 2012). Others have reported the involvement of magnesium balance with nephrocalcinosis (Reeves et al., 1993). Since the loop of Henle does not morphologically mature until PND 28 (Frazier, 2017; Brown et al., 2016), the first week of feeding 5K96 to F1 cohorts (PND 21 – PND 28) may render that animal susceptible to nephrocalcinosis. The basophilic tubules associated with 5K96, reported herein, can appear microscopically similar to, and be easily confused with, chronic progressive nephropathy (CPN), an exceedingly common finding in rats. After all, both are characterized by basophilic tubules. But in CPN, prominent features also include peritubular basement membranes, tubular dilatation, intraluminal protein, hyaline casts, and
Fig. 2. 5K96-Induced Basophilic Tubules. Basophilic tubules (moderate) in the kidney of F1 control female rat fed 5K96 diet. (A) The kidneys were characterized by multifocal collections of basophilic tubules limited to the cortex. These tubules were occasionally dilated (arrowhead). 15 x, H&E. (B) The basophilic tubules had crowded nuclei with slight thickening of basement membranes (arrows). The tubular lumina occasionally had pink intracellular proteinaceous fluid. There is minimal mononuclear cell infiltration into the interstitium associated with affected tubules. 33 x, H&E. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
and more selective for phosphate) (Meloan and Puchtler, 1985; Rungby et al., 1993). Mineralization was consistently at an incidence of 100% in the F1 females, and commonly graded as moderate to marked, with a group mean severity as high as 3.3. The term “basophilic tubules” was a morphologic diagnosis that was characterized by multifocal discrete clusters of tinctorially abnormal tubules in the outer cortex (Fig. 2). The main feature of this lesion was multifocal clusters of tubules lined by epithelial cells having deeply basophilic cytoplasm and crowded nuclei. Other less common features included prominent tubular basement membranes and dilated tubular lumina. Rarely, there was intraluminal protein and interstitial mononuclear inflammatory cell infiltration. The outer cortical location and appearance of the affected tubules were most consistent with that of distal convoluted tubules. The proximal convoluted tubules in the cortex and those tubules within the medulla (loops of Henle and collecting ducts) appeared normal microscopically. The segments of loops of Henle at the corticomedullary junction were commonly effaced by mineral. The incidence of basophilic tubules ranged from 85 to 93% in F1 females, commonly at moderate severity, and the average group severity was up to 1.85. In general, the degree of basophilic tubules paralleled the degree of mineralization, but this correlation was not invariable. The mean kidney weights, blood urea nitrogen (BUN) and serum creatinine values for the four DART studies are provided in Table 2. In all studies, these values were within the Charles River-Ashland historical control range of the study means. While the focus of this publication is to report renal lesions in control animals fed 5K96, these diet-induced lesions were exacerbated in the treated groups of animals. In particular there was test article-related 469
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Table 1 Incidence and severity of renal Lesionsa in control F0 and F1 rats from four DART studies.
Table 2 Mean kidney weights, urea nitrogen, and serum creatinine values in control F0 and F1 rats from four DART studies. F0 Female
Study 1a Study 2 Study 3 Study 4 Historical Control1
Study 1 Study 2 Study 3 Study 4 HistoricalControl1
F0 Males
Age
Kidney Weight (g)
Urea Nitrogen (mg/ dL)
Creatinine (mg/dL)
Kidney Weight (g)
Urea Nitrogen (mg/ dL)
Creatinine (mg/dL)
19–21 wks 19–21 wks 19–21 wks 19–21 wks 19–21 wks
1.8 2.0 2.0 2.2 2.1 (1.6–2.5)
14.4 16.3 18.2 15.4 15.5 (12.2–20.2)
0.4 0.4 0.5 0.5 0.4 (0.4–0.5)
3.0 3.6 3.7 3.8 3.7 (2.8–4.6)
11.7 13.1 14.8 12.0 14.5 (9.0–17.5)
0.4 0.4 0.4 0.4 0.3 (0.3–0.5)
Age
F1 Female Kidney Weight (g)
Urea Nitrogen (mg/ dL)
Creatinine Values (mg/ dL)
F1 Males Kidney Weight (g)
Urea Nitrogen (mg/ dL)
Creatinine Values (mg/ dL)
12 wks 12 wks 12 wks 12 wks 9–12 wks
1.7 2.0 2.0 2.0 1.9 (1.4–2.3)
14.3 14.4 17.1 14.2 15.3 (11.0–20.3)
0.4 0.4 0.4 0.4 0.3 (0.2–0.5)
2.6 3.3 3.4 3.3 2.9 (2.0–3.9)
12.5 11.8 14.0 13.7 13.3 (9.4–17.2)
0.3 0.3 0.4 0.4 0.3 (0.2–0.4)
1
Group mean values for historical control is provided. The numbers in the parentheses refer to either the range of 2 standard deviations from the mean (kidney weights) or range of the study means (urea nitrogen and creatinine). a The control group size for each study was 25 animals per group per sex.
pathophysiologic differences between these lesions. CPN is a lesion of older male rats, while the 5K96-induced basophilic tubules are more prominent in young females (< 12 weeks of age). While both CPN and basophilic tubules are mitigated with a soy-based diet, the protective
inflammatory cell infiltration (Hard et al., 2013). These additional features were minor and variable in the 5K96-induced basophilic tubules reported herein. Not only are there distinct morphologic features between CPN and 5K96-induced basophilic tubules, there are distinct 470
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studies. If 5K96 diet is included in the study design, special consideration may be needed to interpret any dose response increase in renal lesions.
Table 3 Composition of soy-free 5K96 diet and conventional 5001 rodent diets.
Protein source Protein (%) Ca (%) P (%) P -non-phytate (%) Ca:P b Magnesium (%)
LabDiet® 5K96
LabDiet® 5001
Soy-Free
Soy – Containing
Casein 19.1 1.05 0.93 0.71 1.29 0.20
Soybean meal 24.1 0.95 0.68 0.42 1.39 0.21
a
Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.fct.2018.09.044. References Breiderhoff, T., Himmerkus, N., Stuiver, M., Mutig, K., Will, C., Meij, I.C., Bachmann, S., Bleich, M., Willnow, T.E., Muller, D., 2012. Deletion of claudin-10 (Cldn10) in the thick ascending limb impairs paracellular sodium permeability and leads to hypermagnesemia and nephrocalcinosis. Proc. Natl. Acad. Sci. Unit. States Am. 109, 14241–14246. Brown, D.L., Walling, B.E., Mattix, M.E., 2016. The urinary system. In: Atlas of Histology of the Juvenile Rat (Parker GA and Picut CA. Elsevier, San Diego, CA, pp. 395–421 (Chapter 13). Camacho, L., Lewis, S.M., Vanlandingham, M.M., Juliar, B.E., et al., 2017. Comparison of endpoints relevant to toxicity assessment in 3 generations of CD1- mice. Food Chem. Toxicol. 94, 39–56. Cooper, S., Latendresse, J.R., Doerge, D.R., Twaddle, N.C., Fu, X., Delclos, K.B., 2006. Dietary modulation of p-nonylphenol-induced polycystic kidneys in male spraguedawley rats. Toxicol. Sci. 91, 631–642. Frazier, K.S., 2017. Review article. Species differences in renal development and associated developmental nephrotoxicity. Birth Defects Res Part B 109, 1243–1256. Hard, G.C., Banton, M.I., Bretzlaff, R.S., et al., 2013. Consideration of rat chronic progressive nephropathy in regulatory evaluations for carcinogenicity. Toxicol. Sci. 132, 268–275. Keenan, K.P., Coleman, J.B., McCoy, C.L., Hoe, C.M., Soper, K.A., Laroque, P., 2000. Chronic nephropathy in ad libitum overfed Sprague Dawley rats and its early attenuation by increasing degrees of dietary (caloric) restriction to control growth. Toxicol. Pathol. 28, 788–798. Latendresse, J.R., Newbold, R.R., Weis, C.C., Delclos, K.B., 2001. Polycystic Kidney Disease Induced in F1 Sprague-Dawley Rats Fed para-Nonylphenol in a Soy-Free, Casein-Containing Diet. Toxicol. Sci. 62, 140–147. Meloan, S.N., Puchtler, H., 1985. Chemical mechanisms of staining methods: von Kossa's technique. What von Kossa really wrote and a modified reaction for selective demonstration of inorganic phosphate. J. Histotechnol. 8, 11–13. Moore, R.J., Reeves, P.G., Veum, T.L., 1984. Influence of dietary phosphorus and sulphaguanidine levels on P utilization in rats. Br. J. Nutr. 51, 453–465. National Institutes of Health, 2007. NTP Technical Report on the Reproductive Dose Range-finding Toxicity Study of Genistein (CAS No. 446-72-0) Administered in Feed to Sprague-Dawley Rats. Public Health Service, U.S. Department of Health and Human Services Toxicity Report Series Number 79. National Institutes of Health, 2010. NTP Technical Report on the Toxicology and Carcinogenesis Study of Ethinyl Estradiol (CAS No. 57-63-6) in Sprague-Dawley Rats. Public Health Service, U.S. Department of Health and Human Services, NIH Publication No, pp. 10–5889. National Research Council (NRC), 2011. Guide for the Care and Use of Laboratory Animals, Eight Edition. National Academies Press, Washington, D.C. https://doi.org/ 10.17226/12910. Office of Economic Cooperation and Development (OECD), 1998. Principles of Good Laboratory Practice (GLP) ENV/MC/CHEM (98)17. OECD Publishing, Paris revised in 1997. Philbrick, D.J., Bureau, D.P., Collins, F.W., Holub, B.J., 2003. Evidence that soyasaponin Bb retards disease progression in a murine model of polycystic kidney disease. Kidney Int. 63, 1230–1239. Puchtler, H., Meloan, S.N., Terry, M.S., 1969. On the history and mechanism of alizarin and alizarin Red S Stains for Calcium. J. Histochem. Cytochem. 17, 110–124. Ranich, T., Bhathena, S.J., Velasquez, M.T., 2001. Protective effects of dietary phytoestrogens in chronic renal disease. J. Ren. Nutr. 11, 183–193. Rao, G.N., 2002. Diet and kidney diseases in rats. Toxicol. Pathol. 30, 651–656. Reeves, P.G., 1997. Components of the AIN-93 diets as improvements in the AIN-76A diet. J. Nutr. 127, 838S–841S. Reeves, P.G., Rossow, K.L., Lindlauf, J., et al., 1993. Development and testing of the AIN93 purified diets for rodents: results on growth, kidney calcification and bone mineralization in rats and mice. J. Nutr. 123, 1923–1931. Rungby, J., Kassem, M., Eriksen, E.F., Danscher, G., 1993. The von Kossa reaction for calcium deposits: sliver lactate staining increases sensitivity and reduces background. Histochem. J. 25, 446–451. Thigpen, J.E., Setchell, K.D., Saunders, H.E., Haseman, J.K., Gran, M.G., Forsythe, D.B., 2004. Selecting the appropriate rodent diet for endocrine disruptor research and testing studies. ILAR J. 45, 401–416. Travlos, G.S., Hard, G.C., Betz, L.J., Kissling, G.E., 2011. Chronic progressive nephropathy in male F344 rats in 90-day toxicity studies: its occurrence and association with renal tubule tumors in subsequent 2-year bioassays. Toxicol. Pathol. 38, 381–389.
a
Contains isoflavones genistein (86.90 ppm), daidzein (50.47 ppm), and coumestral (6.88 ppm) (CamachoLewis et al., 2017) b Calculated as Calcium:total Phosphorus. However, in monogastric animals, phytate phosphorus is less “available” following digestion than non-phytate phosphorus. The higher the non-phytate phosphorus, the more P that is available, thus lowering the effective Ca:P ratio (Moore et al., 1984; Reeves et al., 1993).
mechanism is likely different. With CPN, the protective effect is linked to the estrogenic isoflavones of soy (Ranich et al., 2001; Philbrick et al., 2003). But since 5K96-induced basophilic tubules are seen primarily in females (with natural circulating levels of estrogen), the protective factor in soy is apparently not the estrogenic isoflavones. It is not clear if 5K96-induced basophilic tubules represent a degenerative or a metabolic change (as is CPN), delayed development (since most tubules in the early postnatal rat up to PND 14 are basophilic) (Brown et al., 2016), or a retrograde nephropathy in response perhaps to mineral imbalance in the thick ascending limb of the loop of Henle. What is clear is there is active morphologic and functional renal development ongoing at the time of early exposure of F1 cohorts to 5K96 (PND 21 – PND 90). Renal morphologic maturation in rats is not complete until PND 28 and functional maturation is not complete until PND 42 (acid base equilibrium is achieved by PND 27; organic anion transporters are mature by PND 35; and stable GFR is achieved by PND 35–42) (Frazier, 2017). These ongoing developmental changes could render the kidney unusually susceptible to certain diets. Regardless of the pathogenesis, these basophilic tubules can mimic toxicity, or they can confound the interpretation or severity of co-existent toxicity. The dose response profiles for basophilic tubules and mineralization, primarily in the F1 female cohorts in two of the four EOGRT studies, do not necessarily suggest direct test article-related toxicity. Given the fact that soy free diets may alter fluid homeostasis, and are associated with a high background incidence of mineralization and basophilic tubules in control F1 animals, these animals are not in homeostasis. It is foreseeable that any additional effect of test article, albeit minor and nonspecific, could exacerbate any pre-existing diet-induced renal lesions, in the same way that some test articles exacerbate CPN (Travlos et al., 2011). 5. Conclusion There is high incidence and severity of basophilic tubules and renal mineralization in studies employing a soy-free 5K96 diet, especially in F1 females fed from weaning up to PND 90. An exaggerated reaction to 5K96 diet in F1 females, when compared to F0 females, for both mineralization and basophilic tubules likely reflects the fact that F1 animals are being exposed to 5K96 diet during morphologic and functional postnatal renal development. Since 5K96-associated renal lesions may complicate interpretation of test article-related renal effects, especially in young F1 or juvenile animals, caution should be exercised before incorporating the 5K96 diet into preclinical DART or juvenile safety
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