Renal Tubulointerstitial Lesions in CBA/J Mice Ulrich H. Rudofsky, BS
Renal tubulointerstitial lesions (RTL) were observed with high frequency in CBA/J mice more than 2 months old. RTL were characterized by interstitial infiltrates of lymphocytes and macrophages in the corticomedullary zone. Multinucleated structures sometimes resembling giant cells were present, and there was destruction of tubules and tubular basement membranes in areas of infiltration. Glomeruli appeared normal. RTL were first seen in 12 of 22 CBA/J mice 2.5 to 3 months old. By the age of 7 to 9 months, 35 of 45 mice were affected, and all 24 mice 12 months old or older had RTL. CBA/J mice had these unique renal lesions whether they were purchased and examined immediately, were obtained as weanlings and reared in our quarters or those of another institution, or were fourth generation descendants of purchased breeders. The propensity to develop RTL has been present in this strain for at least 2 years. RTL were not observed in C57BL/6J mice housed for 14 months with affected animals or in a survey of CBA/HUmc, C57BL/6J, A/j, BALB/cJ, or C3H/HeJ mice. Immunofluorescent examination of CBA/J kidneys appears to rule out antitubular basement membrane autoantibodies or immune complexes in the pathogenesis of RTL. (Am J Pathol 92: 333-348, 1978)
DURING RECENT STUDIES on the renal morphology of long-lived actinomvcin-D-treated (NZB/W)F1 mice,1'2 we observed renal tubulointerstitial lesions (RTL) in some untreated 14-month-old CBA,J control mice. We have investigated the prevalence of RTL in CBA/J mice and the associated histologic changes. The lesions were characterized by mononuclear cell infiltrates in the corticomedullar- zone and destruction of tubules and tubular basement membrane (TBM), and thev did not resemble known spontaneous or induced renal abnormalities in mice.34 Materials and Methods Mice
The histories of the mice used in this studs are summarized in Table 1. lost male and female CBA J mice were obtained directly from the Jackson Laboratory, Bar Harbor. Nte.. and either killed immediately or maintained for varying periods in our quarters. Some CBA J mice were kindly donated by Dr. D. A. Lawrence, Albany NMedical College. These animals were the offspring or descendants of purchased breeders and were killed at arrisval. CBA HUmc mice were obtained from NMiss June Smith, Department of Laboratory From the Nesw York State Kidnes Disease Institute, Disision of Laboratories and Research. Nes'ork State Department of Health, Albany, Nesw York. Supported in part by NIH Research Grant AM 18158-07. aswarded by the National Institute of Arthritis, Mletabolism and Digestise Diseases. PHS DHE\\' Accepted for publication \March 28. 1978. Address reprint requests to Mr. Ulrich H. Rudofsky, Senior Research Scientist, News York State Kidnev Disease Institute, Empire State Plaza Laboratories, Albans, NY 12201. 0002-9440/78/081 0-0333$01 .00 333
334
American Journal of Pathology
RUDOFSKY
Table 1-Histories of the 177 Mice Examined
Age when autopsied
Group
Strain
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
CBA/J
17 18 19 20 21
CBA/HUmc
22 23
C57BLI6J
24
BALB/cJ
25 26
C3H/HeJ
(mo) 1½
1
1 2 1 3 2'½ 3
10 5 5 6 6 10 14 4 10 12 5 5 4
71/2 9
7S-8½12 71/2 7 12 14-18 14-18 >22
A/J
No. in group
11 4
Birth date 4/77 7/77 8/77 9/77 6/77 7/77
7/7 10/76 12/76 1/77 2/77 3/77 5/76
7/75 7/75 -75 7/77
Bred
AMC JL JL JL AMC AMC JL JL
AMC JL JL JL JL JL JL JL
UM UM UM JL JL
2 7 11 7-9 >12
8 5 3 10 6
3/77 10/76 1/77 -/75
7-9 >12
10 5
-/75-76
JL JL
>12
3
-/75
JL
1
5 10
10/77 3/77
JL JL
8-9
1/77
Reared
AMC JL JL JL AMC
AMC JL DLR from 2 mo AMC JL JL JL DLR from 7-9 mo DLR from 2-3 mo DLR from 2-3 mo DLR; exact dates of birth or arrival not known UM UM UM JL DLR; exact dates of birth or arrival not known JL DLR; exact dates of birth or arrival not known DLR; exact dates of birth or arrival not known JL JL
AMC, Albany Medical College; JL, Jackson Laboratory; DLR, Division of Laboratories and Research; UM, University of Minnesota
Medicine and Pathology, University of Minnesota. Other strains (C57BL 6J, A,, J, BALB cJ, and C3H/HeJ) were also purchased from the Jackson Laboratory.
Lght All mice were killed bv cervical dislocation or by exsanguination under CO2 anesthesia. Pieces of kidnev, and in some instances pieces of heart, liver, lung, spleen and small intestine, were fixed in 10% phosphate-buffered formalin or in Tellvesniczky's fixative. Care was taken to obtain complete transverse and/or sagittal sections. The tissues were processed routinely, and sections 3- to 4-M thick were stained with hematoxylin and eosin (H&E) bv conventional procedures. Some sections of renal tissue were stained with silver methenamine (Jones) and NMasson's trichrome. Von Kossa's stain was used to detect calcium deposits and amvloid was sought with Congo red.
TUBULOINTERSTITIAL NEPHRITIS
Vol. 92, No.2 August 1978
335
The degree of RTL was estimated as follows: 4+ lesions involved most of the inner zone of the cortex and outer zone of the medulla; 3+ lesions were large and confluent but only about half as extensive as those scored 4+. Lesions were classed as 2 + or 1 + w-hen there were only focal mononuclear cell infiltrates in the corticomedullarv zone w-ith mild to moderate involvement of some tubules. Immunofluorescent Micoscopy
Pieces of kidney tissue were frozen in isopentane cooled in dry ice and were prepared and stained as previously described.5 Fluorescein-conjugated antiserums to mouse IgG. Ig\M. and IgA (M1eloy Laboratories, Springfield, V-a.) and mouse C3 (Cappel Laboratories. Downington, Pa.) were diluted for optimal staining by titration on kidney sections from a (NZB W)TF mouse with glomerulonephritis. The renal basement membranes were stained by indirect immunofluorescence with antiserum obtained from guinea pigs with autoimmune renal tubulointerstitial disease as previously described." Other Procedures Bladder urines were tested for protein, hemoglobin, glucose, and ketone mvith Labstix
reagent strips (Ames Company, Elkhart, Ind.). Blood urea nitrogen (BUN) was estimated on Azostix reagent strips (Ames) or on Urographs (General Diagnostics. Morris Plains. N.J.).
Results Developnent of RTL in CBA/J Mice
The prevalence of RTL in CBA/J mice is summarized in Tables 2 and 3. Lesions were found in 71 of 112 CBA/J mice. While no significant histologic abnormalities were clearlv apparent in the kidneys of 1- to 2month-old mice, 55% of the 2.5- to 3-month-old mice were affected, and all animals 12 months old or older had RTL. These abnormalities were seen regardless of whether the animals were purchased and killed immeTable 2-Selective Occurrence of RTL in CBA/J Mice Born Between 1975 and 1977 and Autopsied at Various Ages (No. with RTLUNo. Examined)* Age (mo)
Strain
1-2
2 1"3
7-9
>12
6/6w
14/14' 4/4' 8/1010
5/513 4/414 11/11's 4/416
0/11 0/102 0/5' 0/54
2/6' 4/107
CBA/H(Umc)
0/817
-
A/J C57BLU6J BALB/cJ C3H/HeJ
-
-
CBA/J
7/1211 2/512
*
-
-
0/52
-
Superscripts refer to groups in Table 1.
0/51 0/1 on 0/1 on
0/10 '
0/31.
0/6& O/5 -0/34 -
RUDOFSKY
336
American Journal of Pathology
Table 3-Severity of RTL in CBA/J Mice of Various Ages
Degree of RTL Age(mo)
Prevalence (%)
0
1+
2+
3+
0 55
21 10 10
3
9
-
-
-
-
1 1
2 1
3
-
29 22
1-2 21/2-3 7-9 >12
78 100
4+
-
diatelv or maintained from 2 to 14 months in our animal quarters. CBA/J mice which wvere offspring of purchased breeders and raised by another investigator also had RTL; one such mouse was a fourth-generation descendant of purchased breeders. Lesions of similar extent developed wvith high frequency in both male and female CBA/J mice (Table 4). The propensity to develop RTL has been present in CBA/J mice for at least 2 years (July 1975 to July 197 ) (Tables 1 and 2). We did not observe these RTL in the other mouse strains wve examined (Table 2) nor in a large number of NZB, NZW, and NZB/NN mice of -arious ages.7 Morholo of RTL
The abnormalities fell into four distinct morphologic groups, and histologic examination of the kidneys of the CBA 'J mice show-ed that severity of RTL increased with age (Table 3). The earliest kidney sections wvere from 1- to 2-month-old CBA J mice. In most of the 21 animals there was no histologic evidence for tubular or interstitial abnormalities. How ever, rare, single necrotic tubular epithelial cells or small foci of interstitial mononuclear cells wvere seen in the corticomedullarv zone of 2 1-month-old and 3 2-month-old mice. Until larger numbers of mice are examined, we consider these findings insufficient evidence for RTL in CBA/J mice this young although such changes w%ere not seen in 8 CBA, 'HU'mc mice of similar age. Table 4-Distribution of RTL in Male and Female CBA/J Mice Age (mo) 1-2
No. with RTL No. examined Percent
212-3
7-9
5-12
0/6
0/15
8/12
4/10
28/38
7/7
15/15
9/9
0
0
67
40
74
100
100
100
Vol. 92, No. 2 August 1978
TUBULOINTERSTITIAL NEPHRITIS
337
By the age of 2.3 or 3 months, mild to moderate RTL were present in 355c% of the CBA/J mice wve examined (Table 3). The lesions were confined to the corticomedullarv zone and consisted of interstitial infiltrates of macrophages and lymphocytes (Figures 1 and 2) and loss of tubules. Occasional tubular cells wvere undergoing mitosis. In some tubules there was sloughing of epithelial cells into the lumen; karyorrhexis w-as seen infrequently. In 7- to 9-month-old CBA J mice, the RTL had extended to much of the inner zone of the cortex and the outer zone of the medulla (Figure 3). No cystic glomeruli were seen. In addition to cellular infiltration, interstitial fibrosis and congestion of peritubular capillaries and small blood v-essels were evident; frank interstitial hemorrhages 'were seen rarely. Papillary necrosis was not observed (Figure 5). Multinucleated structures wvere the most striking feature in the lesions in CBA 'J mice aged 7 months or older (Figures :3A, 3D. 4A. and 4B). Some of them resembled multinucleated giant cells and contained as many as 20 nuclei (Figures 3D and 4B) but no mitotic figures. Others seemed to be related to damaged tubules, although they were not surrounded by basement membrane. Congestion of intratubular capillaries and small vessels was common. Silver methenamine staining showed that in areas of severe damage the tubular basement membrane was attenuated, disrupted, or absent; fluorescent tests on frozen kidney sections with antiserum to renal basement membranes confirmed this finding. Identification of many cells in the affected areas was difficult. Although the morphology of macrophages and lymphocytes w-as typical at times. a large number of cells wvere epithelioid in appearance and presumably- arose from tubules or macrophages or both (Figures 2B, 3D, 4B, and 4C). A second problem of identification was with cells undergoing mitosis. An increase in the number of mitotic figures was observed in tubules with fairly intact epithelium (Figure 3C), wvhile the majority of the proliferating cells appeared in areas of tubular destruction. In some cases they wvere clearly part of the infiltrate; in other instances these single proliferating cells appeared to be of tubular origin (Figures 3C and 4C). In CBA J mice 12 months old or older RTL were morphologically similar to those seen at 7 to 9 months. Of 6 mice 18 to 22 months old, 1 had severe bilateral hvdronephrosis and 1 had severe glomerular sclerosis. In 3 of these old kidneys, cystic glomeruli Xwere prominent, and there were also infiltrates in the outer cortex. Cholesterol crxstals wvere present in the renal medulla and papilla of most of these mice; amyloid could not be demonstrated with Congo red. No deposits of Ig or C3 Xwere seen along the renal tubular basement
338
RUDOFSKY
American Joumal of Paoogy
membranes in mice with RTL. However, mesangial deposits were present in all glomeruli. Histologic renal abnormalities were also observed in the strains of mice other than CBA/J. None of these 65 mice developed RTL, but they all had age-dependent glomerular changes such as increased mesangial matrix, increased glomerular size, and mild hypercellularity. One 23-monthold C57BL/6J mouse had severe glomerular sclerosis with minimal involvement of tubules. In 9 of 10 8-month-old C3H/HeJ female mice, focal calculi were seen in the corticomedullary zone and these were associated with minimal mononuclear cell infiltrates as described by Dunn 3; such deposits were absent in CBA/J kidneys with RTL. Ottw Os
Mortality could not be followed in animals with RTL in the present series since they were killed at arbitrary intervals. Even in mice with severe lesions, BUN determinations did not indicate renal impairment (Table 5). BUN was mildly elevated (45 mg/dl) in only 2 of the animals tested: a CBA/J mouse with severe hydronephrosis and RTL and a CBA/HUmc mouse with no histologic renal abnormalities. Urinalyses at autopsy on the animals listed in Table 5 were unremarkable (protein, trace to 1+; glucose and hemoglobin, 0; ketone, 0 to trace). In animals with RTL, the heart, lung, liver, spleen, and intestine appeared normal compared with other mice of similar age. Discussion Our observations show that for at least 2 years there has been a high frequency of renal tubular and interstitial lesions in CBA/J mice. Approximately 70% of the mice between 2.5 and 22 months of age were affected whether they were purchased and examined immediately, were obtained as weanlings and raised in our quarters or those of another institution, or were descendants of purchased breeders. Prevalence and severity increased with age. The CBA/HUmc, C57BL/6J, A/J, BALB/cJ, and C3H/ HeJ mice examined did not have RTL. The pathologic features of the lesions have not been described before in this or other mouse strains.3' Most tubular lesions in mice are presumably the consequence of severe glomerulonephritis like that in NZB/W mice 9 or the new MRL and BSBX strains.10 Such tubular and interstitial lesions do not resemble those in CBA/J mice, a strain that does not develop glomerulonephritis. RTL of CBA/J mice differ from chemically induced lesions, such as those induced by chloroform in C3H mice,11 and from inherited nephronophthisis in CBA/CaH mice.U2
TUBULOINTERSTITIAL NEPHRITIS
Vol. 92, No. 2 August 1978
339
Table 5-Blood Urea Nitrogen Determination on Mice With and Wifthout RTL Strain CBA/J
Age (mo)
No. in group
RTL degree
BUN (mg/dl)
1
0 0 1+ 3+ 4+ 4+
21 (15-30)18(10-25) 15 15
18
9 2 1 1 6 2
2 7 11
8 5 3
0 0 0
19 (10-45)t
8
11
0
10
7-9
CBA/HUmc
C57BIJ6J
18(10-25)
38 (30-45)t
13(10-15)
13(10-15)
* Mean (range) t One animal had a BUN of 45 mg/dl and severe hydronephrosis in addition to RTL $ One animal had a BUN of 45 mg/di without histologic evidence of renal disease; all others
had values of 10 to 20 mg/dl.
Whether RTL are caused by noxious or contagious agents or are the result of an inborn disease susceptibility or an inherited metabolic disorder is not known. Our observations indicate that other inbred mice do not have such renal lesions. Since we did not observe RTL in C57BL/6J mice which were housed with affected animals for 14 months in our animal quarters, it seems that this affliction is not readily transmitted to another strain. Preliminary data indicate that these lesions are present in CBA/J offspring raised in a colony separated from the original stock bv four generations. The metabolic effect of these renal lesions is not known. It is astonishing that mice with 3+ to 4+ RTL seem overtly normal, when most of the descending straight limbs of proximal convoluted tubules and Henle's loops are severely disrupted (Figure 3). Proximal convoluted tubules and glomeruli in the outer cortex do not seem to be affected by severe damage in the corticomedullary zone. Further histologic studies are necessary to account for this unexpected long-term maintenance of renal function despite the severity of morphologic abnormalities. Some of the CBA/J mice did not have RTL by 7 to 9 months of age. It is not known whether they are resistant, did not come into contact with the causative agent, or would have developed RTL later. Most mice we examined at 12 to 22 months of age had severe RTL. There is substantial evidence that antitubular basement membrane autoantibodies or immune complexes can cause certain types of RTL in experimental animals 6.13 and in humans,15 but the absence of both Ig and C3 in this area appears to rule out such a mechanism in the pathogenesis
340
RUDOFSKY
American Journal of Pathology
of the lesions in CBA/J mice. However, direct experimental data on the involvement of cell-mediated immunitv in renal tubulointerstitial disease other than transplant rejection are inconsistent.1" RTL in CBA/J mice consist of mononuclear cell infiltrates, but further experiments are necessarv to determine if RTL are an expression of an autoaggressive or autoimmune reaction. The presence of RTL in an inbred mouse strain is of special interest since it mav be a model for the studv of inherited susceptibility to certain forms of tubulointerstitial renal disease. The prevalence of these lesions in CBA/J mice must be considered when they are used for studies of aging and for renal disease experiments. References 1. Gabrielsen AE, Rudofsky UH: Kidnevs of old Dactinomvcin treated NZB NZW' (B/W) and untreated CBA and CK7BL,/6 mice. Fed Proc 36:1296, 1977 2. Rudofsky UH, Urizar RE, Gabrielsen AE, Simmons AD, Olsen CT: Dactinomvcin treatment of murine lupus erythematosus. I. Renal disease and longevity. (Submitted for publication) 3. Dunn TB: Some observations on the normal and pathologic anatomy of the kidney of the mouse. J Natl Cancer Inst 9:285-301, 1949 4. Dunn TB: Renal disease of the mouse. Pathology of Laboratory Rats and Mice. Edited by E Cotchin, FJC Roe. Oxford, Blackwell Scientific Publications, Ltd., 1967, pp 149-179 5. Sommer VNM, Rudofsky UH, Gabrielsen AE: Immune complexes in skin of NZB NZW mice. Clin Exp Immunol 22:461-467, 1973 6. Steblay RW, Rudofskv UH: Transfer of experimental autoimmune renal cortical tubular and interstitial disease in guinea pigs by serum. Science 180:966-968, 1973 7. Rudofskv UH: Unpublished observations 8. Dunn TB: Personal communication 9. Helver BJ, Howie JB: Renal disease associated with lupus erythematosus tests in a cross-bred strain of mice. Nature 197:197, 1963 10. Murphy EF, Roths JB: A single gene model for massive lvmphoproliferation with autoimmunitv in new mouse strain MRL. Fed Proc 36:1246, 1977 11. Deringer NMK. Dunn TB, Heston WE: Results of exposure of strain C3H mice to chloroform. Proc Soc Exp Biol M1ed 83:474-479, 1953 12. Lyon NME, Hulse EV: An inherited kidnev disease of mice resembling human nephronophthisis. J Med Genet 8:41-48, 1971 13. Andres GA, McCluskey RT: Tubular and interstitial renal disease due to immunologic mechanisms. Kidney Int 7:271-289, 1975 14. Heptinstall RH: Interstitial nephritis: A brief review. Am J Pathol 83:214-236, 1976
Acknowledgments The author wishes to express his gratitude to Dr. Thelma B. Dunn for her encouragement to pursue this study, to Dr. William Kaufmann for his advice, and to Messrs. Robert L. Diluith. Louis L. Esposito, Andrew D. Simmons, and Carl J. Eriole for their expert technical support. Ms. Debra Hotaling provided excellent secretarial assistance.
Vol. 92, No. 2 August 1978
TUBULOINTERSTITIAL NEPHRITIS
[llustrations folow]
341
Figure 1-Corticomedullary zone of an unaffected 7-month-old CBA/J mouse. Histology is similar to that seen in other strains. (Silver methenamine, x 256)
A-Mononuclear cell infiltrates are present in the outer zone of the medulla and, to a lesser extent, in the inner zone of the cortex. There is loss of tubular architecture. Although this is a considerable lesion, the overall extent on crossB-At higher magnification, most of the infiltrating cells are lymphocytes section was 2+. and macrophages; neutrophils are not conspicuous. Tubules are in various stages of deterioration. (Silver methenamine; A, x 256; B, x 625) Figure 2-RTL in a 3-month-old CBA/J mouse.
Figure 3-Composite of the typical morphology of RTL in a 7-month-old female CBA/J mouse. A-Low-power view depicting normal outer cortex, mildly affected inner cortex, and severe involvement of outer medulla. Multinucieated structures resembling giant cells are seen (arrows). B-Glomerular histology appears normal for this age. C-Mononuclear cells surround proximal tubules of the inner cortex. Mitotic figure is seen in a tubular Dcell (lower arrow), while the origin of another dividing cell (upper arrow) is doubtful. Multinucleated structure, disrupted tubules, interstitial infiltrates, and cellular proliferation (arrow) are seen. (Silver methenamine; A, x 156; B, x 469; C, x 256; D, x 625)
Figure 4-Severe RTL in a 9-month-old CBA/J mouse. A-Mononuclear infiltrates are present in cortex and medulla. Numerous multinucleated structures are seen (arrows). B and C-Higher magnification shows typical disarray of tubules, which makes the identification of cells difficult. Mitotic figures appear to be originating from infiltrating cells as well as tubules (arrows). (A, H&E, X 156; B, silver methenamine, x 1063; C, H&E, X 1326)
Figure 5-RTL in a 7-month-old mouse. Despite severe RTL (left), the renal papilla appears normal. (Silver methenamine, x 156)
1
41t
3
4
0rn'': " t
PM'
WqId, 4
J, I
Ak
ir t
jc%
i.
.i: 0
.a .
t
. t
00'
.. .
,
iA W
v-*%
'.
v -. .4
Renal tubulointerstitial lesions (RTL) were observed with high frequency in CBA/J mice more than 2 months old. RTL were characterized by interstitial infiltrates of lymphocytes and macrophages in the corticomedullary zone. Multinucleated structures sometimes resembling giant cells were present, and there was destruction of tubules and tubular basement membranes in areas of infiltration. Glomeruli appeared normal. RTL were first seen in 12 of 22 CBA/J mice 2.5 to 3 months old. By the age of 7 to 9 months, 35 of 45 mice were affected, and all 24 mice 12 months old or older had RTL. CBA/J mice had these unique renal lesions whether they were purchased and examined immediately, were obtained as weanlings and reared in our quarters or those of another institution, or were fourth generation descendants of purchased breeders. The propensity to develop RTL has been present in this strain for at least 2 years. RTL were not observed in C57BL/6J mice housed for 14 months with affected animals or in a survey of CBA/HUmc, C57BL/6J, A/j, BALB/cJ, or C3H/HeJ mice. Immunofluorescent examination of CBA/J kidneys appears to rule out antitubular basement membrane autoantibodies or immune complexes in the pathogenesis of RTL. (Am J Pathol 92: 333-348, 1978)
DURING RECENT STUDIES on the renal morphology of long-lived actinomvcin-D-treated (NZB/W)F1 mice,1'2 we observed renal tubulointerstitial lesions (RTL) in some untreated 14-month-old CBA,J control mice. We have investigated the prevalence of RTL in CBA/J mice and the associated histologic changes. The lesions were characterized by mononuclear cell infiltrates in the corticomedullar- zone and destruction of tubules and tubular basement membrane (TBM), and thev did not resemble known spontaneous or induced renal abnormalities in mice.34 Materials and Methods Mice
The histories of the mice used in this studs are summarized in Table 1. lost male and female CBA J mice were obtained directly from the Jackson Laboratory, Bar Harbor. Nte.. and either killed immediately or maintained for varying periods in our quarters. Some CBA J mice were kindly donated by Dr. D. A. Lawrence, Albany NMedical College. These animals were the offspring or descendants of purchased breeders and were killed at arrisval. CBA HUmc mice were obtained from NMiss June Smith, Department of Laboratory From the Nesw York State Kidnes Disease Institute, Disision of Laboratories and Research. Nes'ork State Department of Health, Albany, Nesw York. Supported in part by NIH Research Grant AM 18158-07. aswarded by the National Institute of Arthritis, Mletabolism and Digestise Diseases. PHS DHE\\' Accepted for publication \March 28. 1978. Address reprint requests to Mr. Ulrich H. Rudofsky, Senior Research Scientist, News York State Kidnev Disease Institute, Empire State Plaza Laboratories, Albans, NY 12201. 0002-9440/78/081 0-0333$01 .00 333
334
American Journal of Pathology
RUDOFSKY
Table 1-Histories of the 177 Mice Examined
Age when autopsied
Group
Strain
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
CBA/J
17 18 19 20 21
CBA/HUmc
22 23
C57BLI6J
24
BALB/cJ
25 26
C3H/HeJ
(mo) 1½
1
1 2 1 3 2'½ 3
10 5 5 6 6 10 14 4 10 12 5 5 4
71/2 9
7S-8½12 71/2 7 12 14-18 14-18 >22
A/J
No. in group
11 4
Birth date 4/77 7/77 8/77 9/77 6/77 7/77
7/7 10/76 12/76 1/77 2/77 3/77 5/76
7/75 7/75 -75 7/77
Bred
AMC JL JL JL AMC AMC JL JL
AMC JL JL JL JL JL JL JL
UM UM UM JL JL
2 7 11 7-9 >12
8 5 3 10 6
3/77 10/76 1/77 -/75
7-9 >12
10 5
-/75-76
JL JL
>12
3
-/75
JL
1
5 10
10/77 3/77
JL JL
8-9
1/77
Reared
AMC JL JL JL AMC
AMC JL DLR from 2 mo AMC JL JL JL DLR from 7-9 mo DLR from 2-3 mo DLR from 2-3 mo DLR; exact dates of birth or arrival not known UM UM UM JL DLR; exact dates of birth or arrival not known JL DLR; exact dates of birth or arrival not known DLR; exact dates of birth or arrival not known JL JL
AMC, Albany Medical College; JL, Jackson Laboratory; DLR, Division of Laboratories and Research; UM, University of Minnesota
Medicine and Pathology, University of Minnesota. Other strains (C57BL 6J, A,, J, BALB cJ, and C3H/HeJ) were also purchased from the Jackson Laboratory.
Lght All mice were killed bv cervical dislocation or by exsanguination under CO2 anesthesia. Pieces of kidnev, and in some instances pieces of heart, liver, lung, spleen and small intestine, were fixed in 10% phosphate-buffered formalin or in Tellvesniczky's fixative. Care was taken to obtain complete transverse and/or sagittal sections. The tissues were processed routinely, and sections 3- to 4-M thick were stained with hematoxylin and eosin (H&E) bv conventional procedures. Some sections of renal tissue were stained with silver methenamine (Jones) and NMasson's trichrome. Von Kossa's stain was used to detect calcium deposits and amvloid was sought with Congo red.
TUBULOINTERSTITIAL NEPHRITIS
Vol. 92, No.2 August 1978
335
The degree of RTL was estimated as follows: 4+ lesions involved most of the inner zone of the cortex and outer zone of the medulla; 3+ lesions were large and confluent but only about half as extensive as those scored 4+. Lesions were classed as 2 + or 1 + w-hen there were only focal mononuclear cell infiltrates in the corticomedullarv zone w-ith mild to moderate involvement of some tubules. Immunofluorescent Micoscopy
Pieces of kidney tissue were frozen in isopentane cooled in dry ice and were prepared and stained as previously described.5 Fluorescein-conjugated antiserums to mouse IgG. Ig\M. and IgA (M1eloy Laboratories, Springfield, V-a.) and mouse C3 (Cappel Laboratories. Downington, Pa.) were diluted for optimal staining by titration on kidney sections from a (NZB W)TF mouse with glomerulonephritis. The renal basement membranes were stained by indirect immunofluorescence with antiserum obtained from guinea pigs with autoimmune renal tubulointerstitial disease as previously described." Other Procedures Bladder urines were tested for protein, hemoglobin, glucose, and ketone mvith Labstix
reagent strips (Ames Company, Elkhart, Ind.). Blood urea nitrogen (BUN) was estimated on Azostix reagent strips (Ames) or on Urographs (General Diagnostics. Morris Plains. N.J.).
Results Developnent of RTL in CBA/J Mice
The prevalence of RTL in CBA/J mice is summarized in Tables 2 and 3. Lesions were found in 71 of 112 CBA/J mice. While no significant histologic abnormalities were clearlv apparent in the kidneys of 1- to 2month-old mice, 55% of the 2.5- to 3-month-old mice were affected, and all animals 12 months old or older had RTL. These abnormalities were seen regardless of whether the animals were purchased and killed immeTable 2-Selective Occurrence of RTL in CBA/J Mice Born Between 1975 and 1977 and Autopsied at Various Ages (No. with RTLUNo. Examined)* Age (mo)
Strain
1-2
2 1"3
7-9
>12
6/6w
14/14' 4/4' 8/1010
5/513 4/414 11/11's 4/416
0/11 0/102 0/5' 0/54
2/6' 4/107
CBA/H(Umc)
0/817
-
A/J C57BLU6J BALB/cJ C3H/HeJ
-
-
CBA/J
7/1211 2/512
*
-
-
0/52
-
Superscripts refer to groups in Table 1.
0/51 0/1 on 0/1 on
0/10 '
0/31.
0/6& O/5 -0/34 -
RUDOFSKY
336
American Journal of Pathology
Table 3-Severity of RTL in CBA/J Mice of Various Ages
Degree of RTL Age(mo)
Prevalence (%)
0
1+
2+
3+
0 55
21 10 10
3
9
-
-
-
-
1 1
2 1
3
-
29 22
1-2 21/2-3 7-9 >12
78 100
4+
-
diatelv or maintained from 2 to 14 months in our animal quarters. CBA/J mice which wvere offspring of purchased breeders and raised by another investigator also had RTL; one such mouse was a fourth-generation descendant of purchased breeders. Lesions of similar extent developed wvith high frequency in both male and female CBA/J mice (Table 4). The propensity to develop RTL has been present in CBA/J mice for at least 2 years (July 1975 to July 197 ) (Tables 1 and 2). We did not observe these RTL in the other mouse strains wve examined (Table 2) nor in a large number of NZB, NZW, and NZB/NN mice of -arious ages.7 Morholo of RTL
The abnormalities fell into four distinct morphologic groups, and histologic examination of the kidneys of the CBA 'J mice show-ed that severity of RTL increased with age (Table 3). The earliest kidney sections wvere from 1- to 2-month-old CBA J mice. In most of the 21 animals there was no histologic evidence for tubular or interstitial abnormalities. How ever, rare, single necrotic tubular epithelial cells or small foci of interstitial mononuclear cells wvere seen in the corticomedullarv zone of 2 1-month-old and 3 2-month-old mice. Until larger numbers of mice are examined, we consider these findings insufficient evidence for RTL in CBA/J mice this young although such changes w%ere not seen in 8 CBA, 'HU'mc mice of similar age. Table 4-Distribution of RTL in Male and Female CBA/J Mice Age (mo) 1-2
No. with RTL No. examined Percent
212-3
7-9
5-12
0/6
0/15
8/12
4/10
28/38
7/7
15/15
9/9
0
0
67
40
74
100
100
100
Vol. 92, No. 2 August 1978
TUBULOINTERSTITIAL NEPHRITIS
337
By the age of 2.3 or 3 months, mild to moderate RTL were present in 355c% of the CBA/J mice wve examined (Table 3). The lesions were confined to the corticomedullarv zone and consisted of interstitial infiltrates of macrophages and lymphocytes (Figures 1 and 2) and loss of tubules. Occasional tubular cells wvere undergoing mitosis. In some tubules there was sloughing of epithelial cells into the lumen; karyorrhexis w-as seen infrequently. In 7- to 9-month-old CBA J mice, the RTL had extended to much of the inner zone of the cortex and the outer zone of the medulla (Figure 3). No cystic glomeruli were seen. In addition to cellular infiltration, interstitial fibrosis and congestion of peritubular capillaries and small blood v-essels were evident; frank interstitial hemorrhages 'were seen rarely. Papillary necrosis was not observed (Figure 5). Multinucleated structures wvere the most striking feature in the lesions in CBA 'J mice aged 7 months or older (Figures :3A, 3D. 4A. and 4B). Some of them resembled multinucleated giant cells and contained as many as 20 nuclei (Figures 3D and 4B) but no mitotic figures. Others seemed to be related to damaged tubules, although they were not surrounded by basement membrane. Congestion of intratubular capillaries and small vessels was common. Silver methenamine staining showed that in areas of severe damage the tubular basement membrane was attenuated, disrupted, or absent; fluorescent tests on frozen kidney sections with antiserum to renal basement membranes confirmed this finding. Identification of many cells in the affected areas was difficult. Although the morphology of macrophages and lymphocytes w-as typical at times. a large number of cells wvere epithelioid in appearance and presumably- arose from tubules or macrophages or both (Figures 2B, 3D, 4B, and 4C). A second problem of identification was with cells undergoing mitosis. An increase in the number of mitotic figures was observed in tubules with fairly intact epithelium (Figure 3C), wvhile the majority of the proliferating cells appeared in areas of tubular destruction. In some cases they wvere clearly part of the infiltrate; in other instances these single proliferating cells appeared to be of tubular origin (Figures 3C and 4C). In CBA J mice 12 months old or older RTL were morphologically similar to those seen at 7 to 9 months. Of 6 mice 18 to 22 months old, 1 had severe bilateral hvdronephrosis and 1 had severe glomerular sclerosis. In 3 of these old kidneys, cystic glomeruli Xwere prominent, and there were also infiltrates in the outer cortex. Cholesterol crxstals wvere present in the renal medulla and papilla of most of these mice; amyloid could not be demonstrated with Congo red. No deposits of Ig or C3 Xwere seen along the renal tubular basement
338
RUDOFSKY
American Joumal of Paoogy
membranes in mice with RTL. However, mesangial deposits were present in all glomeruli. Histologic renal abnormalities were also observed in the strains of mice other than CBA/J. None of these 65 mice developed RTL, but they all had age-dependent glomerular changes such as increased mesangial matrix, increased glomerular size, and mild hypercellularity. One 23-monthold C57BL/6J mouse had severe glomerular sclerosis with minimal involvement of tubules. In 9 of 10 8-month-old C3H/HeJ female mice, focal calculi were seen in the corticomedullary zone and these were associated with minimal mononuclear cell infiltrates as described by Dunn 3; such deposits were absent in CBA/J kidneys with RTL. Ottw Os
Mortality could not be followed in animals with RTL in the present series since they were killed at arbitrary intervals. Even in mice with severe lesions, BUN determinations did not indicate renal impairment (Table 5). BUN was mildly elevated (45 mg/dl) in only 2 of the animals tested: a CBA/J mouse with severe hydronephrosis and RTL and a CBA/HUmc mouse with no histologic renal abnormalities. Urinalyses at autopsy on the animals listed in Table 5 were unremarkable (protein, trace to 1+; glucose and hemoglobin, 0; ketone, 0 to trace). In animals with RTL, the heart, lung, liver, spleen, and intestine appeared normal compared with other mice of similar age. Discussion Our observations show that for at least 2 years there has been a high frequency of renal tubular and interstitial lesions in CBA/J mice. Approximately 70% of the mice between 2.5 and 22 months of age were affected whether they were purchased and examined immediately, were obtained as weanlings and raised in our quarters or those of another institution, or were descendants of purchased breeders. Prevalence and severity increased with age. The CBA/HUmc, C57BL/6J, A/J, BALB/cJ, and C3H/ HeJ mice examined did not have RTL. The pathologic features of the lesions have not been described before in this or other mouse strains.3' Most tubular lesions in mice are presumably the consequence of severe glomerulonephritis like that in NZB/W mice 9 or the new MRL and BSBX strains.10 Such tubular and interstitial lesions do not resemble those in CBA/J mice, a strain that does not develop glomerulonephritis. RTL of CBA/J mice differ from chemically induced lesions, such as those induced by chloroform in C3H mice,11 and from inherited nephronophthisis in CBA/CaH mice.U2
TUBULOINTERSTITIAL NEPHRITIS
Vol. 92, No. 2 August 1978
339
Table 5-Blood Urea Nitrogen Determination on Mice With and Wifthout RTL Strain CBA/J
Age (mo)
No. in group
RTL degree
BUN (mg/dl)
1
0 0 1+ 3+ 4+ 4+
21 (15-30)18(10-25) 15 15
18
9 2 1 1 6 2
2 7 11
8 5 3
0 0 0
19 (10-45)t
8
11
0
10
7-9
CBA/HUmc
C57BIJ6J
18(10-25)
38 (30-45)t
13(10-15)
13(10-15)
* Mean (range) t One animal had a BUN of 45 mg/dl and severe hydronephrosis in addition to RTL $ One animal had a BUN of 45 mg/di without histologic evidence of renal disease; all others
had values of 10 to 20 mg/dl.
Whether RTL are caused by noxious or contagious agents or are the result of an inborn disease susceptibility or an inherited metabolic disorder is not known. Our observations indicate that other inbred mice do not have such renal lesions. Since we did not observe RTL in C57BL/6J mice which were housed with affected animals for 14 months in our animal quarters, it seems that this affliction is not readily transmitted to another strain. Preliminary data indicate that these lesions are present in CBA/J offspring raised in a colony separated from the original stock bv four generations. The metabolic effect of these renal lesions is not known. It is astonishing that mice with 3+ to 4+ RTL seem overtly normal, when most of the descending straight limbs of proximal convoluted tubules and Henle's loops are severely disrupted (Figure 3). Proximal convoluted tubules and glomeruli in the outer cortex do not seem to be affected by severe damage in the corticomedullary zone. Further histologic studies are necessary to account for this unexpected long-term maintenance of renal function despite the severity of morphologic abnormalities. Some of the CBA/J mice did not have RTL by 7 to 9 months of age. It is not known whether they are resistant, did not come into contact with the causative agent, or would have developed RTL later. Most mice we examined at 12 to 22 months of age had severe RTL. There is substantial evidence that antitubular basement membrane autoantibodies or immune complexes can cause certain types of RTL in experimental animals 6.13 and in humans,15 but the absence of both Ig and C3 in this area appears to rule out such a mechanism in the pathogenesis
340
RUDOFSKY
American Journal of Pathology
of the lesions in CBA/J mice. However, direct experimental data on the involvement of cell-mediated immunitv in renal tubulointerstitial disease other than transplant rejection are inconsistent.1" RTL in CBA/J mice consist of mononuclear cell infiltrates, but further experiments are necessarv to determine if RTL are an expression of an autoaggressive or autoimmune reaction. The presence of RTL in an inbred mouse strain is of special interest since it mav be a model for the studv of inherited susceptibility to certain forms of tubulointerstitial renal disease. The prevalence of these lesions in CBA/J mice must be considered when they are used for studies of aging and for renal disease experiments. References 1. Gabrielsen AE, Rudofsky UH: Kidnevs of old Dactinomvcin treated NZB NZW' (B/W) and untreated CBA and CK7BL,/6 mice. Fed Proc 36:1296, 1977 2. Rudofsky UH, Urizar RE, Gabrielsen AE, Simmons AD, Olsen CT: Dactinomvcin treatment of murine lupus erythematosus. I. Renal disease and longevity. (Submitted for publication) 3. Dunn TB: Some observations on the normal and pathologic anatomy of the kidney of the mouse. J Natl Cancer Inst 9:285-301, 1949 4. Dunn TB: Renal disease of the mouse. Pathology of Laboratory Rats and Mice. Edited by E Cotchin, FJC Roe. Oxford, Blackwell Scientific Publications, Ltd., 1967, pp 149-179 5. Sommer VNM, Rudofsky UH, Gabrielsen AE: Immune complexes in skin of NZB NZW mice. Clin Exp Immunol 22:461-467, 1973 6. Steblay RW, Rudofskv UH: Transfer of experimental autoimmune renal cortical tubular and interstitial disease in guinea pigs by serum. Science 180:966-968, 1973 7. Rudofskv UH: Unpublished observations 8. Dunn TB: Personal communication 9. Helver BJ, Howie JB: Renal disease associated with lupus erythematosus tests in a cross-bred strain of mice. Nature 197:197, 1963 10. Murphy EF, Roths JB: A single gene model for massive lvmphoproliferation with autoimmunitv in new mouse strain MRL. Fed Proc 36:1246, 1977 11. Deringer NMK. Dunn TB, Heston WE: Results of exposure of strain C3H mice to chloroform. Proc Soc Exp Biol M1ed 83:474-479, 1953 12. Lyon NME, Hulse EV: An inherited kidnev disease of mice resembling human nephronophthisis. J Med Genet 8:41-48, 1971 13. Andres GA, McCluskey RT: Tubular and interstitial renal disease due to immunologic mechanisms. Kidney Int 7:271-289, 1975 14. Heptinstall RH: Interstitial nephritis: A brief review. Am J Pathol 83:214-236, 1976
Acknowledgments The author wishes to express his gratitude to Dr. Thelma B. Dunn for her encouragement to pursue this study, to Dr. William Kaufmann for his advice, and to Messrs. Robert L. Diluith. Louis L. Esposito, Andrew D. Simmons, and Carl J. Eriole for their expert technical support. Ms. Debra Hotaling provided excellent secretarial assistance.
Vol. 92, No. 2 August 1978
TUBULOINTERSTITIAL NEPHRITIS
[llustrations folow]
341
Figure 1-Corticomedullary zone of an unaffected 7-month-old CBA/J mouse. Histology is similar to that seen in other strains. (Silver methenamine, x 256)
A-Mononuclear cell infiltrates are present in the outer zone of the medulla and, to a lesser extent, in the inner zone of the cortex. There is loss of tubular architecture. Although this is a considerable lesion, the overall extent on crossB-At higher magnification, most of the infiltrating cells are lymphocytes section was 2+. and macrophages; neutrophils are not conspicuous. Tubules are in various stages of deterioration. (Silver methenamine; A, x 256; B, x 625) Figure 2-RTL in a 3-month-old CBA/J mouse.
Figure 3-Composite of the typical morphology of RTL in a 7-month-old female CBA/J mouse. A-Low-power view depicting normal outer cortex, mildly affected inner cortex, and severe involvement of outer medulla. Multinucieated structures resembling giant cells are seen (arrows). B-Glomerular histology appears normal for this age. C-Mononuclear cells surround proximal tubules of the inner cortex. Mitotic figure is seen in a tubular Dcell (lower arrow), while the origin of another dividing cell (upper arrow) is doubtful. Multinucleated structure, disrupted tubules, interstitial infiltrates, and cellular proliferation (arrow) are seen. (Silver methenamine; A, x 156; B, x 469; C, x 256; D, x 625)
Figure 4-Severe RTL in a 9-month-old CBA/J mouse. A-Mononuclear infiltrates are present in cortex and medulla. Numerous multinucleated structures are seen (arrows). B and C-Higher magnification shows typical disarray of tubules, which makes the identification of cells difficult. Mitotic figures appear to be originating from infiltrating cells as well as tubules (arrows). (A, H&E, X 156; B, silver methenamine, x 1063; C, H&E, X 1326)
Figure 5-RTL in a 7-month-old mouse. Despite severe RTL (left), the renal papilla appears normal. (Silver methenamine, x 156)
1
41t
3
4
0rn'': " t
PM'
WqId, 4
J, I
Ak
ir t
jc%
i.
.i: 0
.a .
t
. t
00'
.. .
,
iA W
v-*%
'.
v -. .4
