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. Author manuscript; available in PMC: 2025 May 14.
Published in final edited form as: Toxicol Pathol. 2011 Feb;39(2):381–389. doi: 10.1177/0192623310388432

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

Greg S Travlos 1, Gordon C Hard 2, Laura J Betz 3, Grace E Kissling 1
PMCID: PMC12077603  NIHMSID: NIHMS2079684  PMID: 21422264

Abstract

The occurrence and severity of spontaneous chronic progressive nephropathy (CPN) in control male F344 rats as well as the frequency of treatment-related CPN exacerbation were histopathologically reevaluated. A series of 43 National Toxicology Program (NTP) 90-day toxicity studies comparing the influence of NIH-07 or NTP-2000 diets was examined. Relationships between the histopathologic findings at 90 days and renal tubule proliferative lesions recorded in subsequent 2-year bioassays for 24 chemicals were statistically analyzed. CPN lesions were observed in 100% of the control male rats regardless of diet, but CPN was more severe in control rats fed NIH-07. Approximately one-third of the 90-day studies demonstrated a treatment-related exacerbation of CPN severity, which was independent of diet. For chemicals that proceeded to 2-year bioassays, all studies with a statistically significant increase in renal tubule tumors (RTT) at 2 years had treatment-related exacerbation of CPN in the 90-day and 2-year studies. These findings indicate that CPN occurs ubiquitously in young male F344 rats and that treatment-related exacerbation of CPN in 90-day studies is a relatively common occurrence, having the potential to be predictive of an increased incidence of RTT in subsequent 2-year bioassays.

Keywords: chronic progressive nephropathy, CPN, NIH-07 and NTP-2000 diets, toxicity and carcinogenicity studies, CPN exacerbation, renal tubule neoplasia

Introduction

Chronic progressive nephropathy (CPN) is a common, spontaneous disease of the rat kidney, and it has been the subject of many detailed reviews (see Hard, Johnson, and Cohen 2009). While all strains of rat used in toxicity and carcinogenicity studies are affected, albino strains such as the F344 and Sprague-Dawley are more inclined to develop the disease. CPN occurs in both sexes but appears with greater frequency and severity in males. Although the etiology of CPN is unknown, numerous factors, mainly physiological, influence the incidence and severity of this spontaneous disease (Hard and Khan 2004). In addition to the male hormone effect (Baylis 1994), caloric restriction and dietary protein content are the most important factors in CPN, with caloric restriction being protective (Keenan et al. 2000) and high protein content increasing incidence and severity (Rao, Edmondson, and Elwell 1993). In 1994, in an effort to reduce various spontaneous lesions observed in toxicity and carcinogenicity studies, including CPN, the National Toxicology Program (NTP) replaced the NIH-07 diet, which contained ~24% protein, with the NTP-2000 diet containing ~14% protein.

The kidney is an important target organ in carcinogenicity bioassays conducted by the NTP, ranking along with liver as one of the most commonly affected sites for tumors in male rats (NTP 2010b), primarily tumors of the renal tubule (RTT). Of 498 NTP 2-year bioassays performed in male rats, 64 (12.9%) demonstrated site-specific tumor occurrence involving renal tubule cells (NTP 2010c, 2010b). However, in female rats, the kidney does not rank in the top 10 site-specific neoplasms, involving only 3.3% (18 of 504) bioassays (NTP 2010b). In contrast, RTT of spontaneous origin in male rats occurs with a relatively low incidence rate of 1.26% (44 of 3,484 animals) in NTP 2-year bioassays where NIH-07 diet was used (NTP 2010a). Female rats fed NIH-07 have an even lower incidence rate of 0.29% (10 of 3,474 animals; NTP 2010a). Based on the NTP historical control animal database, it appears that dietary manipulations designed to mitigate CPN and its potential long-term effects also lowered the incidence of spontaneous RTT. NTP 2-year bioassays using the NTP-2000 diet have continued to demonstrate a gender difference for the occurrence of spontaneous RTT, with the incidence rate decreasing to 0.62% (8 of 1,294 animals) and 0.16% (2 of 1,236 animals) for male and female F344 rats, respectively (NTP 2010a). Along with the alpha2u-globulin (α2u-g) nephropathy mode of action (Hard et al. 1993; Swenberg and Lehman-McKeeman 1999), a potential explanation for the predilection of the kidney as a common site for chemically induced neoplasms in male rats and the gender difference in RTT incidence might be chemical exacerbation of CPN (Hard, Johnson, and Cohen 2009).

Since doses used in NTP 2-year bioassays are selected based on data obtained from 90-day subchronic toxicity studies, renal histopathology in subchronic studies might provide insight for kidney lesions occurring in the chronic studies. The present study was therefore designed to (1) assess the frequency of CPN occurring in control F344 male rats in 90-day studies, (2) determine the frequency of chemical exacerbation of CPN occurring in 90-day studies, and (3) examine possible relationships between treatment-induced exacerbation of CPN at 90 days and microscopic findings (including occurrence of RTT) at 2 years. Accordingly, histopathologic reevaluation of CPN and other kidney lesions in control and treated male F344 rats was conducted for a series of NTP 90-day studies. The findings from this assessment were then compared with the 2-year outcomes recorded for those chemicals that proceeded to 2-year bioassays. Since diet can have a profound biological effect on CPN, the effects of the two standardized, open formula diets, NIH-07 and NTP-2000, were compared.

Materials and Methods

Study Evaluation

Kidneys from forty-three 90-day toxicity studies (Table 1) conducted by the NTP including control and highest-exposure treated male F344 animals were reevaluated for incidence and severity of CPN and incidence of non-CPN lesions (1,090 rats in total). Since the NTP had changed to the NTP-2000 diet, which contained less protein and more fiber and fat than the original NIH-07 diet, 90-day studies were selected as close to the time of the diet switch in 1994 as possible; all were conducted from 1991 to 2001. Of the 43 studies, 22 used the NIH-07 diet and 21 the NTP-2000 diet. Both diets were produced and supplied by Zeigler Brothers, Inc. (Gardners, PA).

Table 1.—

Ninety-day NTP toxicity studies (along with diet and route of administration) in which kidney histopathology was reevaluated.a

Chemical Name (NIH-07 Diet) Route Chemical Name (NTP-2000 Diet) Route
Coconut oil diethanolamine (TR 479) Dermal Pentaerythritol triacrylate (GMM 4) Dermal
Dicyclohexylcarbodiimide (GMM 9) Dermal Trimethylolpropane triacrylate (GMM 3) Dermal
Diisopropylcarbodiimide (TR 523) Dermal p-tertiary-butylcatechol (TS 70) Feed
Lauric acid diethanolamide (TR 480) Dermal Chromium picolinate monohydrate (TR 556) Feed
Oleic acid diethanolamide (TR 481) Dermal Citral (TR 505) Feed
Anthraquinone (TR 494) Feed Transcinnamaldehyde (TR 514) Feed
Benzophenone (TS 61, TR 533) Feed Acrolein (TS 48) Gavage
p,p-dichlorodiphenyl sulfone (TR 501) Feed Allyl alcohol (TS 48) Gavage
trans-1,2-dichloroethylene (TS 55) Feed Butanal oxime (TS 69) Gavage
1,1,2,2-tetrachloroethane (TS 49) Feed 2,4-decadienal (TS 76) Gavage
o-chloroaniline (TS 43) Gavage Elmiron (TR 512) Gavage
Oxymetholone (TR 485) Gavage Estragole (TS 82) Gavage
3,3,4,4-tetrachloroazobenzene (TS 65) Gavage Formamide (TR 541) Gavage
3,3,4,4-tetrachloroazoxybenzene (TS 66) Gavage 2,4-hexadienal (TR 509) Gavage
Methylene blue trihydrate (TR 540) Gavage Alpha-methyl styrene (TR 543) Inhalation
2-butoxyethanol (TR 484) Inhalation Decalin (TR 513) Inhalation
Indium phosphide (TR 499) Inhalation Divinylbenzene (TR 534) Inhalation
Isobutene (TR 487) Inhalation Propylene glycol mono-t-butyl ether (TR 515) Inhalation
1-nitropyrene (TS 34) Inhalation Stoddard solvent IIC (TR 519) Inhalation
Benzyltrimethylammonium chloride (TS 57) Water Dibromoacetic acid (TR 537) Water
Urethane/water (TS 52) Water Sodium dichromate dihydrate (TS 72, TR 546) Water
Urethane/alcohol (TS 52) Water
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a

For the chemicals listed, the number in parentheses is the National Toxicology Program (NTP) report number; TS = NTP Toxicity Report. Series, TR = NTP Technical Report Series, GMM = NTP Genetically Modified Model Report Series.

For chemicals with a 2-year carcinogenicity bioassay, CPN and non-CPN kidney lesions were also reevaluated in the 90-day exposure groups that most closely approximated the highest-exposure group used in the 2-year study (2-year–equivalent dose). The 90-day kidney lesions were compared with the 2-year histopathologic findings recorded in the NTP Technical Reports. For six of the chemicals (diisopropylcarbodiimide, isobutene, chromium picolinate, decalin, divinylbenzene and propylene glycol monobutyl ether), the 2-year–equivalent dose was also the highest-exposure dose in the 90-day study.

The studies had been conducted in accordance with the standard protocols established by the NTP (Chhabra et al. 1990). In general, for the 90-day studies, groups of 10 male and 10 female F344 rats (Taconic Laboratory Animals and Services, Germantown, NY) had been either untreated or exposed to the vehicle used (control groups) or exposed to one of five doses of a chemical for 13 consecutive weeks. All rats were approximately 4 to 7 weeks old at study commencement. Kidneys (along with other tissues for histopathology) had been fixed in 10% neutral-buffered formalin, and 5-μm sections were stained with hematoxylin and eosin (H&E). For the 2-year carcinogenicity bioassays, groups of 50 male and 50 female F344 rats had been untreated or exposed to a vehicle (controls) or exposed to one of two or three dose levels of chemical for 104 consecutive weeks. Histopathological data for kidneys from the 2-year bioassays were taken directly from the NTP Technical Reports, and kidney sections from those studies were not reevaluated in this investigation.

Assessment of kidney histopathology in the 90-day studies was performed by one of the authors (G.C.H.). H&E sections of both kidneys (one transverse, one sagittal in most cases) from all control and high-exposure male animals of the forty-three 90-day studies were examined in their entirety in an up-and-down battlement pattern for evidence of CPN or treatment-related lesions. Male rats were chosen for review because they are more affected by CPN than female rats (Gray 1977). For chemicals tested in 2-year carcinogenicity bioassays, kidney sections from the 90-day-dose groups that approximated (identical or closest to) the highest dose used in the 2-year study were examined in the same manner.

Grading of CPN

A specialized system for grading CPN was used, based on in-depth study of the pathogenesis of this spontaneous disease over many years (Hard and Khan 2004; Hard, Johnson, and Cohen 2009). The earliest stage of CPN was identified as single basophilic tubules with thickened basement membranes (Figure 1a) usually in the cortex, foci of tubules with the same characteristics, or tubules dilated with a hyaline proteinaceous cast in the medulla (Figure 1b). The severity of CPN was graded on a scale ranging from 0 to 8 (Hard and Khan 2004). In this system, 0 represented no lesions observed in either kidney section, 1 = minimal (≤5 lesions per both sections), 2 = mild (6–15 lesions), 3 = low-moderate (16–30 lesions), 4 = mid-moderate (30–60 lesions), 5 = high-moderate (focal lesions/casts too numerous to count), 6 = low-severe, 7 = high-severe, 8 = end-stage. Grades 1 through 5 represented increasing numbers of focal lesions. Grade 6 represented a stage at which the focal lesions began to coalesce into areas. In grade 7, the majority of the cortical parenchyma was affected, while in grade 8, little or no normal parenchyma remained, representing an end-stage kidney.

Figure 1.—

Figure 1.—

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(a) A series of basophilic tubule profiles representing a single nephron affected by chronic progressive nephropathy (CPN), extending in a linear fashion (arrows) from the cortex, through the outer stripe of the outer medulla, and down to the inner stripe of the outer medulla junction. Diagnosis of CPN could be based on any of the profiles indicated by an arrow. However, this particular lesion with its discontinuous profiles would be scored as only one focus of CPN. (b) A series of hyaline (or colloid) casts in the inner stripe of the outer medulla represents the turns of a single tubule. Hyaline casts in the inner stripe of the outer medulla or inner medulla are characteristic of CPN and indicate more proximal (cortical) CPN involvement of the same nephron. The presence of an isolated cast of this type (arrows), without apparent association with a basophilic focus in the cortex, or linear basophilic tubules in the outer stripe of the outer medulla, would be counted as one CPN lesion.

Statistical Analyses

The Wilcoxon rank-sum test (Hollander and Wolfe 1973) was used to test differences in effect between the two diets on CPN severity in 90-day control groups; the data were also analyzed separately for each route. The Kruskal-Wallis analysis of variance followed by Dunn’s multiple comparison’s procedure (Hollander and Wolfe 1973) was used to determine whether routes of chemical exposure were statistically different for CPN severity in the control groups; the data were analyzed separately for each diet. The association of CPN severity with other endpoints of renal change (e.g., absolute and relative kidney weights) was determined using Spearman’s rank correlation coefficient (Hollander and Wolfe 1973) on the mean values reported for each study; control and high-dose groups were analyzed separately. A two-sided, 0.05 level of significance was used for these analyses.

To assess the effects of chemical treatment on CPN, the mean severity grade for the control, high-, and 2-year–equivalent dose groups were calculated for each chemical. The difference between the control and high-dose group or the control and 2-year–equivalent means was found. A difference of ≥0.5 in severity was considered to be an increase (i.e., exacerbation) in CPN, while a difference of ≤−0.5 in severity was considered a decrease (i.e., reduction); values between −0.5 and 0.5 were considered no change. While this was an arbitrary decision point, the cutoff was chosen based on experience and used to reconcile statistically significant differences that would otherwise be overlooked. Jonckheere’s trend (Hollander and Wolfe 1973) and Wilcoxon’s tests supported the conclusions based on this cutoff. An exact chi-square test (Hollander and Wolfe 1973) was performed to compare trend calls (i.e., exacerbated, reduced, or no change in CPN) between diets and route of exposure. It was also used for comparing CPN trend calls in the 2-year–equivalent dose of the 90-day studies with the neoplastic and nonneoplastic findings reported in the NTP 2-year studies.

Kidney tumor incidences from the NTP 2-year studies were reanalyzed for this investigation using the poly-3 test (Bailer and Portier 1988) and a hybrid test that incorporated historical control data (Peddada, Dinse, and Kissling 2007); kidney hyperplasia incidences were analyzed using the poly-3 test. Incidences of RTT or hyperplasias were considered increased if they were significantly elevated above the control group in at least one dose group or if there was a significant increasing trend with dose at one-sided p ≤ 0.05. In keeping with NTP practices, historical control groups were comprised of data from control animals in studies using the same route of exposure and conducted within the most recent 5 years prior to the study of interest. The poly-3 test has been in use for NTP 2-year studies since 1999. The hybrid statistics are now being routinely calculated on all new NTP 2-year studies and will be included in NTP technical reports subject to acceptance by the NTP Board of Scientific Counselors.

Results

Studies Evaluated

The chemicals evaluated in this review of 90-day studies are listed in Table 1 according to diet type and route of exposure. There were approximately equal numbers of studies for the two diets (22 for NIH-07 and 21 for NTP-2000), but the distribution of route of exposure varied between diets. For example, the NIH-07 diet had a relatively equal number of studies for the different routes of exposure (n = 5, 5, 5, 4, and 3 for dermal, feed, gavage, inhalation, and drinking water routes of exposure, respectively). In contrast, the NTP-2000 diet had a higher number of gavage studies (8) and a lower number of dermal studies (2) compared with the NIH-07 diet.

CPN in Control Male Rats

Regardless of diet or route of exposure, early lesions of CPN were found in 100% of control male rats in all of the forty-three 90-day studies (n = 220 and 230 animals for NIH-07 and NTP-2000, respectively). Severity grades ranged from 1 to 4, with 35.8%, 55.6%, 7.3%, and 1.3% of all control rat kidneys categorized as minimal, mild, low-moderate, and mid-moderate, respectively. However, the grade of CPN severity in control rats was significantly higher (p < 0.0001) in studies giving the NIH-07 diet (mean severity ± SE = 2.0 ± 0.04) versus the NTP-2000 diet (1.5 ± 0.03). Of the control males fed NTP-2000, 54.4% had CPN categorized as grade 1 (minimal) compared with 16.4% for NIH-07, 0.4% of the males fed NTP-2000 had CPN categorized as grade 3 (low-moderate) compared with 14.6% for NIH-07, and only the NIH-07 diet had control male kidneys (2.7%) with a severity grade of 4 (mid-moderate). The inhalation route of exposure had the lowest grade of CPN among the control male rats, irrespective of diet. As CPN severity increased, absolute and relative kidney weights increased (data not shown).

Effects of Chemical Treatment on CPN Severity in 90-Day Studies

Regardless of diet (NIH-07 or NTP-2000), there were no differences in treatment-induced exacerbation or reduction of CPN in the high-exposure groups (Table 2). In addition, route of exposure was unrelated to exacerbation or reduction in CPN severity in the high-dose groups (data not shown). Fifteen chemicals, representing 35.7% of the total study set (6 NIH-07 and 9 NTP-2000), had an exacerbation of CPN severity in the high-exposure males, whereas 14 chemicals, representing 33.3% of the study set (9 NIH-07 and 5 NTP-2000), had a reduction of CPN severity, in some cases to grade 0. For 13 chemicals (31.0% of the study set; 6 NIH-07 and 7 NTP-2000), there was no change in CPN severity. For seven of the studies, the highest-exposure groups could not be evaluated for CPN, either due to low survival (benzophenone, urethane in water, dicyclohexylcarbodiimide, acrolein, and butanal oxime) or overriding non–CPN-related kidney lesions (3,3′,4,4′-tetrachloroazoxybenzene and indium phosphide). Consequently, for six of these seven chemicals, the next-to-highest dose group was examined and included in the statistical analysis. Indium phosphide was excluded because of a lack of an appropriate dose where CPN could be distinguished from chemically induced nephropathy.

Table 2.—

Number of 90-day studies with chemical-related exacerbation, reduction, or no change in chronic progressive nephropathy (CPN).a

Exacerbated Reduced No Change Total Studies
NIH07 dietb 6 9 6 21
NTP2000 diet 9 5 7 21
Combined 15 (35.7%) 14 (33.3%) 13 (31.0%) 42
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a

Chemical-related change in CPN was determined by ±0.5 differences in mean severity grades between the control and high-exposure groups.

b

For the NIH-07 diet, one study (indium phosphide) was not evaluated for chemical-related change in CPN due to overriding non-CPN–related kidney lesions.

CPN in 90-Day Studies and RTT Incidences at 2 Years

Twenty-four of the 43 chemicals evaluated in the 90-day studies proceeded to 2-year bioassays (Table 3), involving an equal number for each of the two diets. Of these 24 chemicals, nine (four NIH-07 and five NTP-2000) demonstrated a treatment-related exacerbation in CPN severity in the 90-day studies at the 2-year–equivalent dose (Table 3); seven of the nine chemicals had an exacerbation of CPN severity reported in the 2-year study. All seven chemicals (three NIH-07 and four NTP-2000) showing exacerbation of CPN in both the 90-day and 2-year studies demonstrated a significantly increased incidence of RTT (representing marginal-only increases in some cases) using the statistical methods described above. In addition, five of the seven chemicals with a significantly increased incidence of RTT had an increased incidence of renal hyperplasia; there was no instance in which increased renal hyperplasia occurred in the absence of increased RTT (Table 3). Four chemicals of the data set were considered by the NTP to be α2u-g inducers, of which three were associated with a significantly increased incidence of RTT (Table 3). There were no apparent associations between increased RTT occurrence and chemicals considered genotoxic by the NTP (Table 3).

Table 3.—

Relationship between chemically-induced chronic progressive nephropathy (CPN) exacerbation and increased incidence of renal tubule tumors/hyperplasia, including NTP-reported genotoxicity and alpha2u-globulin (α2u-g) association.

CPN Chemical Name Tumorsa Hyperplasiab Genotoxicityc α2u-gd
Exacerbated in 90-day and 2-year studies Anthraquinone Yes No +
Benzophenone Yes Yes
Decalin Yes Yes +
Divinylbenzene Yes Yes
Oxymetholone Yes No
Propylene glycol mono-t-butyl ether Yes Yes + +
Stoddard solvent IIC Yes Yes +
Exacerbated in 90-day study only Citral No No +
p,p-dichlorodiphenyl sulfone No No +
Not exacerbated in either study 2-butoxyethanol No No
Chromium picolinate monohydrate No No
Coconut oil diethanolamine No No +
Dibromoacetic acid No No +
Diisopropylcarbodiimide No No +
Elmiron No No
Formamide No No
2,4-hexadienal No No +
Indium phosphide No No
Isobutene No No
Lauric acid diethanolamide No No
Methylene blue trihydrate No No +
Oleic acid diethanolamide No No
Sodium dichromate dihydrate No No +
Transcinnamaldehyde No No +
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a

Yes = a statistically significant trend and/or pairwise difference from the controls by the poly-3 test or by the hybrid test incorporating historical controls (p ≤ 0.05); no = statistical analyses were not significant (p > 0.05).

b

Yes = a statistically significant trend and/or pairwise difference from the controls by the poly-3 test (p ≤~0.05); no = statistical analyses were not significant (p > 0.05).

c

+ = Chemicals considered by NTP to be genotoxic in one or more short-term assays.

d

+ = Chemicals considered by NTP to be candidates for acting via the α2u-g pathway.

Since there was an association (p < 0.0001) between treatment-related exacerbation in CPN severity in the 90-day studies and increased incidence of RTT at 2 years, performance measures of the 90-day CPN findings as an indicator of increased RTT were calculated (Table 4). Based on this set of 24 studies, exacerbation of CPN at 90 days was a sensitive (100%) and specific (88%) indicator of increased RTT incidence at 2 years, predicting the presence or absence of increased RTT in a 2-year bioassay with 78% or 100% accuracy, respectively.

Table 4.—

Calculation of performance measures for exacerbation of chronic progressive nephropathy (CPN) in 90-day studies to indicate the increased incidence of renal tubule tumors (RTT) in 2-year studies.

2-year bioassays Total
Number of studies with RTTa Number of studies with no RTT
Number of studies with exacerbated CPNb in the 90-day study 7 2 9
Number of studies with no CPN exacerbation in the 90-day study 0 15 15
Total 7 17 24
Performance measures
Sensitivity (probability of exacerbated CPN with RTT) 7/7 = 100%
Specificity (probability of no exacerbated CPN with no RTT) 15/17 = 88%
+ Predictive value (probability of RTT with exacerbated CPN) 7/9 = 78%
− Predictive value (probability of no RTT with no exacerbated CPN) 15/15 = 100%
Efficiency (probability that the 90-day and 2-year results agree) 22/24 = 92%
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a

Increased incidence of RTT was determined by a statistically significant trend and/or pairwise difference from the controls by the poly-3 test or by the hybrid test incorporating historical controls (p ≤ 0.05).

b

Chemical-related exacerbation in CPN was determined by a ≥0.5 difference in the mean severity grades between the control group and the dose group in the 90-day study that was identical to, or most closely approximated, the top dose used in the 2-year bioassay.

Other Treatment-Related Lesions

Apart from CPN effects, the most common treatment-related lesion in male rats of the forty-three 90-day studies was hyaline droplet accumulation in cortical proximal tubules (eight studies), usually accompanied by granular cast formation at the junction of outer and inner stripes of the outer medulla (six of the eight studies; Table 5). Of the twenty-four 90-day studies that proceeded to a 2-year bioassay, five had hyaline droplet accumulation (anthraquinone, citral, decalin, propylene glycol monobutyl ether, and Stoddard solvent IIC). The diagnosis of hyaline droplet accumulation/granular cast formation in the 90-day studies was positively associated with an increased incidence of RTT in the 2-year bioassays (p = 0.015). Although correspondingly specific (94%) for indicating increased RTT incidence at 2 years as CPN exacerbation (88%), hyaline droplet accumulation/granular cast formation at 90 days was not as sensitive (57%) as CPN exacerbation (100%).

Table 5.—

Chemical-related kidney lesions other than chronic progressive nephropathy (non-CPN) observed in the histopathological reevaluation of forty-three 90-day NTP studies.a

All 90-day studiesb 90-day studies proceeding to 2-year bioassaysc
Number of studies evaluated 43 24
Number of studies with lesions 19 7
Lesion
 Hyaline droplet accumulationd 8 5
 Tubular pigment 7 1
 Granular casts or precursors 6 5
 Mineralizatione 3 1
 Glomerulopathy 2
 Nephrosis 2
 Cytoplasmic vacuolization 1
 Linear papillary mineralization 1
 Papillary necrosis 1
 Pyelonephritis 1
 Tubular hyperplasia 1
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a

Data are reported as number of studies with the lesion.

b

Kidney sections from control and high-exposure male rats were evaluated for evidence of treatment-related non-CPN lesions.

c

Kidney sections from control males and treated male rats from the 90-day study whose dose approximated the highest dose used in the 2-year bioassay were evaluated for evidence of treatment-related non-CPN lesions.

d

Hyaline droplets were consistent with α2u-globulin in five of eight studies and four of five studies in the “all” and “2-year” study sets, respectively.

e

Mineralization was intratubular and located at the outer stripe of the medulla/inner stripe of the medulla junction.

Discussion

CPN is regarded as a disease of old rats; however, the findings in this microscopic review of kidneys from 43 NTP 90-day studies provide confirmation that early stages of the disease can be observed in relatively young F344 males. Thus, lesions of CPN were detected at minimal to low-moderate levels of severity in 100% of the control male rats at approximately 4 to 5 months of age. Dixon, Heider, and Elwell (1995) reported similar findings for control male F344 rats from NTP 90-day studies (approximately 91% of the males reviewed); conversely, other reports have indicated no or low (<10%) incidence of CPN for male rats of approximately the same age as the animals reviewed here (Coleman et al. 1977; Maeda et al. 1985).

An explanation for this difference is most likely the specialized 0 to 8 grading scheme used to assess CPN in our reevaluation. Conventional schemes are based on estimating the percentage of parenchyma affected by CPN, using a qualitative 0 to 4 or occasionally a 0 to 5 grading scale (Coleman et al. 1977; Rao, Edmondson, and Elwell 1993). In one of these grading schemes, the lowest (minimal) grade represented involvement of less than 20% of the cortex and outer medulla by CPN, consisting of several small foci of renal tubular cell degeneration/regeneration and occasional tubules containing proteinaceous casts (Rao, Edmondson, and Elwell 1993). In the present study, however, a semiquantitative method was used based on a numerical count of focal lesions for the low grades (Hard and Khan 2004). For investigative purposes, incremental grading of CPN severity on a consistently measurable stage of disease progression is more suitable for elucidating subtle effects and detecting statistical differences than using a subjective assessment of percentage area of kidney involved. For example, a severity grade of 1 typically used in a conventional scheme may cover severity grades 1 to 4 in the expanded 0 to 8 grading system. Furthermore, for the chemicals evaluated in this review, the entity of CPN was not reported or scored by the NTP as a diagnosis in the 90-day studies, reflecting differences in grading methodology, which precluded any comparison between the NTP assessments at 90 days and the results reported here. Thus, the semiquantification of very low numbers of tubule lesions increased the sensitivity of the grading method, enabling identification of CPN-affected animals at the earliest histomorphologic stage. It should be emphasized that this 0 to 8 grading system is a specialized, labor-intensive research tool used for CPN-focused assessments only. It is not practical or recommended for use in routine histopathological evaluations of kidney sections in toxicity studies.

In this study, the type of diet had a significant effect on the severity of CPN in control male animals. While the incidence of CPN was not altered, the severity was 25% lower in control male animals on the lower protein diet (NTP-2000) compared with the higher protein diet (NIH-07). This is consistent with the beneficial kidney effects previously reported for the NTP-2000 diet. Rao, Edmondson, and Elwell (1993) demonstrated that decreasing the dietary protein concentration from 23% to 15% reduced the severity of CPN in male rats at 2 years by approximately 50%. Our study shows that the reduction in CPN severity associated with feeding a low-protein diet commences early (at least by 4 to 5 months of age). Furthermore, there was a positive correlation between CPN severity and absolute and relative kidney weights regardless of diet. Such a correlation suggests that the presence of CPN lesions augments the weight of the organ, even at an early stage of lesion development. This would be in keeping with the fact that end-stage CPN kidneys are increased in size, in contrast with end-stage kidneys from various causes in humans, where typically there is shrinkage of the organ (Hard, Johnson, and Cohen 2009).

No previous reports have estimated the frequency with which chemically induced CPN exacerbation in safety assessment studies occurs. Treatment-related exacerbation of CPN was observed in approximately one-third of the 43 studies evaluated here. Conversely, treatment-related reduction of CPN occurred at a similar rate. Thus, treatment-related alterations in CPN, either exacerbation or reduction, can be a common finding (70%) in 90-day studies. Whereas exacerbation of CPN is regarded as an adverse event, reduction of CPN may likely be a response to treatment-related stress. The structures of chemical agents known to exacerbate CPN are diverse, and a mechanism for the enhancing effect is not known (Doi et al. 2007). Since the expression of CPN can be modified by nutritional factors (reviewed in Hard and Khan 2004), a difference in rates of treatment-related CPN exacerbation/reduction between the NTP-2000 and NIH-07 diets might have been expected in this study, but there was no evidence for such a dietary role. Thus, chemicals destined to exacerbate CPN apparently did so whether a low- or high-protein diet was used.

Our investigation showed a strong association between CPN exacerbation at 90 days and subsequent RTT occurrence. After statistical recalculation, all of the seven chemicals demonstrating an increased incidence of RTT in the 2-year bioassay (see Table 3) had a treatment-related exacerbation in CPN at both 90 days and 2 years. In addition, five of these same seven chemicals had an increased incidence of tubule hyperplasia, a lesion considered by NTP (NTP 2006) and others (Hard 1987; Lipsky and Trump 1988; Dietrich and Swenberg 1991; Nogueira, Cardesa, and Mohr 1993) to be on a developmental continuum with RTT. There was no increase in hyperplasia for any of the remaining 24 chemicals taken to a 2-year endpoint. Consequently, the data on tubule hyperplasia supported the association between CPN and increased incidence of RTT (see Table 3).

The notion that increases in CPN severity in chronic studies can be associated with increased frequency of RTT is not new. Anver et al. (1982) reported pathology data from two stocks of untreated Sprague-Dawley rats held into senescence, indicating that the stock with the higher incidence of spontaneous renal adenoma also had the highest severity of CPN over comparable age groups and life span. In other work, it has been noted that an increase in RTT frequently occurred in dose groups exhibiting chemical exacerbation of CPN (Montgomery and Seely 1990). In their characterization of the utility of multiple-section sampling and evaluation of the kidney for carcinogenicity studies, Eustis et al. (1994) used, as one of their criteria for study selection, a chemical-related increase in the average severity of CPN. This criterion was included because, in their experience, slight or marginal increases in renal tubule hyperplasia and/or adenoma often accompanied chemical-related exacerbation of CPN. Furthermore, they noted that a chemical-related increased severity of nephropathy was seen in male rats for every study in which step-section evaluations were useful in establishing a relationship between chemical administration and increased incidences of renal proliferative lesions. More recently, Seely et al. (2002) demonstrated a slight, but statistically significant, association between increased severity grades of CPN in male F344 rats with RTT compared with age-matched animals without tumors. A statistically significant positive association of RTT with an advanced stage of CPN has now been shown for carcinogenicity studies of hydroquinone (Hard et al. 1997), ethyl benzene (Hard 2002), and quercetin (Hard et al. 2007). The findings reported here add further support to the work cited above.

A novel aspect of our data is that chemically induced exacerbation of CPN at 90 days may be a predictive indicator of RTT occurrence at 2 years. In a recent investigation sponsored by International Life Sciences Institute seeking biological endpoints predictive of a tumorigenic outcome (Boobis et al. 2009), a kidney weight increase at 13 weeks was the only finding that was consistently associated with renal tumors. Although our results suggest that chemically induced exacerbation of CPN at 90 days also has that potential to predict a subsequent increase in RTT (see Table 4), there are some caveats to be considered. First, the selection criteria used for this evaluation resulted in the identification of studies that, in general, had been performed on industrial chemicals or environmental/food contaminants; no studies involving drugs in development, small molecules, dietary supplements, or physical agents were included. Second, the seven chemicals associated with RTT may not have represented a broad enough range of modes of action operative in renal carcinogenesis (Hard 1998; Lock and Hard 2004). Although anthraquinone is regarded as a genotoxic chemical (NTP 2005), it was of one of five chemicals that induced hyaline droplet accumulation at 90 days. Hyaline droplet nephropathy is known to be accompanied by CPN exacerbation (Hard et al. 1993), and this association may have skewed the results in favor of demonstrating a correlation between subchronic CPN exacerbation and chronic RTT occurrence. In addition, as CPN is characterized by a highly elevated proliferative rate (Short, Burnett, and Swenberg 1989), a synergistic interaction between hyaline droplet nephropathy and advanced CPN resulting in a marginal increase in RTT incidence is plausible (Lock and Hard 2010). Finally, female rats were not included in the study. Therefore, at this stage, our findings should be regarded as pertinent to the male rat only. As rat CPN has no counterpart in humans (Hard et al. 2009), RTT arising in association with chemically induced CPN exacerbation should have no relevance for species extrapolation in human risk assessment.

Acknowledgments

This research was supported, in part, by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences, and by NIH contract N01ES55547. The authors wish to thank Drs. Kim Weber and Louise Fitzgerald for their assistance in the preliminary organization of Table 1.

Abbreviations:

α2u-g

alpha2u-globulin

CPN

chronic progressive nephropathy

H&E

hematoxylin and eosin

NTP

National Toxicology Program

RTT

renal tubule tumor

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