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Histopathology of subacute renal lesions in mice induced by streptozotocin
Exp Toxic Pathol 1995; 47: 485--491 Gustav Fischer Verlag lena
Laboratory of Biomedical Science, Department of Veterinary Medical Science, Faculty of Agriculture, The University of Tokyo, Japan
Histopathology of subacute renal lesions in mice induced by streptozotocin SHIN-ICHI ITAGAKI, ETSUKO NISHIDA, MIN-JAE LEE and KUNIO DOl With 12 figures and 1 table Received: December 1, 1994; Accepted: April 25, 1995 Address for correspondence: Dr. SHIN-IeHI ITAGAKI, Laboratory of Biomedical Science, Department of Veterinary Medical Science, Faculty of Agriculture, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan. Key words: Hyperglycemia; Streptozotocin, renal lesions; Renal lesions, streptozotocin; Kidney, streptozotocin.
Summary Streptozotocin (SZ) was inoculated intraperitoneally to male and female mice of ICR and BALB/c strains in a different way of administration (A: single injection and B: consecutive 5 days-injection) and subacute renal changes were examined light and electron microscopically 8 weeks after SZ-administration. The following changes were detected: (1) reduction in the rate of male-type Bowman's capsules in male mice, (2) karyocytomegaly of proximal tubular epithelial cells, and (3) dilatation of distal tubules. Tubular changes were detected with high incidence in A (males and females) and B groups (males) ofICR strain.
Introduction Streptozotocin (SZ) has been used as a useful tool for either the induction of insulin-dependent diabetes mellitus or the investigation of its complications in laboratory rodents (CORTES et al. 1987; IWASE et al. 1986; JAKOBSON et al. 1987; LIKE et al. 1978; SPANHEIMER et al. 1988). SZ is also known as a carcinogenic agent to induce renal adenomas and carcinomas when administered to rats and mice (ARISON and FEUDALE 1967; HARD 1985; HORTON et al. 1977; KAZUMI et al. 1978; MAUER et al. 1974; OKAWA and DOl 1983; RAKIETEN et al. 1968). Although urinary tubules are affected in the subacute phase (CHIECO et al. 1993; HONJO et al. 1986; KANEDA et al. 1992; RASCH 1984), the details of the lesion and its relation to finally formed renal tumors are still unclear. In this connection, comparative bioassays conducted in rats with alloxan, a similar diabetogenic agent, suggest that neoplastic growth is due to a direct toxic effect of SZ on kidney cells and not to the induced diabetes (MAUER et al. 1974). In this study, using male and female mice of two strains and two ways of administration, we tried to esta-
blish the best experimental system for further investigations of SZ-induced renal tumors and clarify the light and electron microscopic features of SZ-induced subacute renal lesions. Pathogenesis of SZ-induced renal lesions in this study will be also discussed.
Material and methods Twenty-five each of male and female mice ofCrj:CD1 (ICR; Charles River Japan Inc., Kanagawa) and BALB/c Cr SIc strains (BALB/c; Japan SLC Inc., Shizuoka) were obtained at 7 weeks of age (initial body weights (B.W.); male ICR: 35.3 ± 2.0 g; female ICR: 26.6 ± 1.8 g; male BALB/c: 25.0 ± 1.3 g; female BALB/c: 20.5 ± 1.0 g). They were housed 5 per polycarbonate cage with wood chips for bedding under controlled conditions (temperature, 23 ± 2°C; humidity, 55 ± 5 %). Food (MF pellets, Oriental Yeast Co., Ltd., Tokyo) and water were available ad libitum. Mice of each strain were divided into 3 groups including the same number of males and females and treated intraperitoneally with SZ at 8 weeks of age as follows; A group (n = 10): single administration of 200 mg/kg B.W. of SZ; B group (n = 10): administration of 50 mg/kg B.W. SZ for 5 consecutive days; C group (n = 5): administration of citrate buffer alone for 5 consecutive days. In other words, all mice were divided into 12 groups (lCRMale (M)-A, -B and -C; ICR-Female (F)-A, -B and -C; BALB/c-M-A, -B and -C; BALB/c-F-A, -B and -C; Table 1). SZ (Sigma Chemical Company, St. Louis, MO) was dissolved in citrate buffer (pH 4.5) immediately before daily treatment. All mice were sacrificed by heart puncture under ether anesthesia 8 weeks after completion of the treatment. Kidneys were fixed in 10 % neutral buffered formalin, and 4 /lm-paraffin sections were cut and Exp Toxic Pathol 47 (1995) 6
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stained with hematoxylin and eosin (HE). Small pieces of the kidney obtained from each mouse were fixed with 2.S % glutaraldehyde, 2.0 % paraformaldehyde in O.OS M phosphate buffer (pH 7.4), postfixed with 1 % osmium tetroxide in the same buffer and processed for electron microscopy. Ultrathin sections were double stained with uranyl acetate and lead citrate, and observed under a JEOL 1200 EX electron microscope (JEOL Co., Ltd., Tokyo). In male mice, it is well known that two types of Bowman's capsules are observed (one is lined with cuboidal to low columnar epithelia and the other with flattened epithelia). The number of each type of Bowman's capsules was counted on HE-stained coronal sections of the kidney (one section per mouse) and the ratio of Bowman's capsules lined with cuboidal to low columnar epithelia to all Bowman's capsules was calculated in all male mice. Values were expressed as mean ± SD and statistical analysis was performed using Student's t-test.
Blood glucose levels were colorimetrically measured on each serum sample using the Glucose C-test kit (Wako Pure Chemical Industries Ltd., Osaka).
Results The incidence of hyperglycemia and histopathological characteristics of renal lesions are summarized in table 1.
Blood glucose levels The average of blood glucose levels was 198.8 ± 24.96 mg/dl in C group (both sex of both strains), and animals which exhibited the blood glucose level more than 300 mg/dl were scored as hyperglycemic. All animals of ICR-M-B and ICR-F-A groups, 80 % of ICR-FB group and 60 % of ICR-M-A and BALB/c-M-A and
Table 1. Incidence of hyperglycemia and histopathological changes in the kidney. Hyperglycemia ICR-MA(10)d BOO)
+(6) -(4) +(10)
C(S)
-(S)
ICR-FAOO) BOO)
Bowman's cap." (%).
40.9 ± 14.3 e *** 71.5 ± 10.6* 24.7 ± 14.9*** 88.6 ± 6.S2
+(0) +(8) -(2)
Proximal tubules b
Distal tubules c
++(4)
++(3), +(2)
+(4)
++(2), +(7)
+(2)
++(4), +(S)
++(1), +(1)
-(5)
C(5) BALB/c-MA(10) B(10)
+(6) -(4) +(6) -(4) -(S)
C(S)
BALB/c-FA(lO) BOO)
+(1) -(9) -(10)
C(S)
-(S)
21.6 ± 8.00*** SS.S ± 17.3* 24.0 ± 7.88*** S1.5 ± 14.6** 81.8 ± 4.48
+(3) +(1)
+(1) +(1)
+(1)
+(1) +(2)
Ratio of Bowman's capsules composed of cuboidal to low columnar epithelial cells to all Bowman's capsules; Karyocytomega1y of proximal tubules (+: moderate, ++: prominent); Dilatation of distal tubules (+: mild to moderate, ++: cyst formation); d Number of parenthesis indicates number of mice; e Values are mean ± S.D. *·**·***Significantly different from C group (*p < O.OS, **p < 0.01, ***p < 0.001). a
b C
486
Exp Toxic Patho1 47 (1995) 6
Fig. 1. Kidney of a mouse in ICR-M-C group. Bowman's capsule is lined with cubodal epithelium. HE. x430. Fig. 2. Kidney of a mouse in ICR-M-A group. Bowman's capsule is lined with flattened epithelium. HE. x430. Fig. 3. Kidney of a mouse in ICR-M-A group. Proximal tubule epithelial cells show moderate karyocytomegaly (arrow-
heads). HE. x430.
Fig. 4. Kidney of a mouse in ICR-M-A group. Proximal tubule epithelial cell shows prominent karyocytomegaly (arrow-
head). HE. x430.
Fig. 5. Kidney of a mouse in ICR-F-A group. Distal tubules show mild to moderate luminal dilatation. A piled-up appearance of epithelial cells (arrowhead) is seen. HE. x21S. Fig. 6. Kidney of a mouse in ICR-F-A group. Distal tubule forms a large cyst. HE. x150.
Exp Toxic PathoI 47 (1995) 6
487
Fig. 7. Cuboidal epithelial cell of Bowman's capsule of a mouse in ICR-M-A group. x4,000. Fig. 8. Flattened epithelial cell (arrowhead) of Bowman's capsule of a mouse in ICR-M-B group. x7,000. -B groups showed hyperglycemia (table 1). In contrast, only one mouse exhibited hyperglycemia in BALB/c-FA and no one in BALB/c-F group (table 1).
Light microscopic findings Light microscopic changes were mainly detected in the renal cortex. Renal corpuscle: Glomeruli of SZ-treated mice showed no prominent changes. Bowman' s capsules of male mice of C group were generally lined with cuboidal to low columar epithelia (fig. 1). On the other hand, in male mice of SZ-treated groups, the number of Bowman's capsules with cuboidal to low columnar epithelia significantly decreased and many Bowman' s capsules were lined with flattened epithelia (fig. 2). This tendency was most prominent in hyperglycemic mice of both strains (table 1). Bowman ' s capsules in female mice were lined with flattened epithelia and no differences were detected among all groups. Proximal tubule: Mild to moderate karyocytomegaly were observed in some epithelial cells in SZ-treated groups except for ICR-F-B and BALB/c-F-B groups (fig. 3). Especially in 40 % of mice of ICR-M-A group, prominet changes were detected in some epithelial cells (fig. 4). Although these features were mainly detected in hyperglycemic mice, similar changes were also observed in non-hyperglycemic BALB/C mice. 488
Exp Toxic Pathol47 ( \995) 6
Distal tubule: Mild to moderate luminal dilatation of distal tubules was observed in hyperglycemic mice of ICR-M and -F and BALB/c-F groups (fig. 5). A piled-up appearance was also seen in some distal tubule epithelia lining the dilated lumen (fig. 5). Furthermore, in some SZ-treated ICR mice, cysts which were supposed to be derived from dilated distal tubules were observed (fig. 6).
Electron microscopic findings Bowman's capsule: The ultrastructure of cuboidal to low columnar cells seen in male mice was similar to that of proximal tubule epithelial cells. Namely, they were characterized by long microvilli, prominent mitochondria, and extensive invaginations of the basolateral plasma membrane (fig. 7). Flattened epithelial cells observed in many SZ-treated male mice had poor organella and their features were similar to those of epithelial cells of Bowman's capsule in female mice (fig. 8). Proximal tubules: Epithelial cells with enlarged nuclei had electron-lucent cytoplasm and poor basal infoldings, and they lacked microvilli on the apical border (fig. 9). Aggregation of mitochondria was observed in some degenerative epithelial cells (fig. 10). Distal tubule: Epithelial cells in moderately dilated tubules had normal organella although the cytoplasm of some cells was elongated and protruded into the lumen (fig. 11). Epithelial cells of cystic tubules lacked micro-
... ..
•
~!1
Fig. 9. Proximal tubule epithelial cell with karyocytomegaly of a mouse in ICR-M-B group. x3,400. Fig. 10. Proximal tubule epithelial cells of a mouse in ICR-M-B group. Aggregation of mitochondria (arrowhead) is seen. x3,400. Fig. 11. Epithelial cells of moderately dilated distal tubule of a mouse in ICR-M-B group. x3,400. Fig. 12. Epithelial cells of cyst-formed distal tubule of a mouse in ICR-M-B group. x3,700.
Exp Toxic Pathol47 (1995) 6
489
villi and basal infoldings, and some of these epithelial cells contained many vacuoles with amorphous contents (fig. 12). Within mitochondria, electron-dense deposits were detected.
Discussion In this study, SZ was inoculated intraperitoneally to male and female mice of ICR and BALB/c strains in a different way of administration. Subacute renal changes were mainly observed in Bowman's capsules, proximal tubules and distal tubules in the cortex in some groups. Tubular changes were detected with high incidence in ICR-M-A and -B and ICR-F-A groups. Prominent karyocytomegaly in proximal tubule epithelial cells was seen especially in ICR-M-A group. These data suggest that the above-mentioned combination of strain of mice, sex and way of inoculation is considered to be best as an experimental system for further investigation of the origin of tumor cells in the kidney of SZ-treated mice. As mentioned above, Bowman's capsules of sexually mature male mice are generally lined with cuboidal to low columnar epithelia. They are called "male-type of Bowman's capsules", and their epithelial cells are suggested to be similar to those of proximal tubules functionally as well as morphologically (LIEBELT 1986). In this study, male-type of Bowman's capsule were seen in approximately 80 % of renal corpuscles in control male mice of both strains. The ratio of male-type Bowman's capsule significantly decreased by SZ-treatment and this tendency was enhanced by hyperglycemia. Diabetic conditions might influence the morphology of male-type epithelial cells probably through continuously increased hydrostatic pressure in the glomerular cavity due to marked and persistent urine production (KUME et al. 1992). However, an other agent is considered to play an additional role in the initiation of this change. Because, the ratio of male-type of Bowman's capsule was statistically lower even in non-hyperglycemic mice than in control mice. Karyocytomegaly of renal tubule epithelial cells is reported in rats following exposure to some renal toxicants and carcinogens such as aflatoxin Bland G l' ochratoxin, nitrosourea, bromodichloromethane, and some heavy metal compounds (MONTGOMERY and SEELY 1990; PAYNE and SAUNDERS 1978; RICHARDSON and WOODARD 1986). Although the pathogenesis is not known, many agents causing karyocytomegaly inhibit mitosis (MONTGOMERY and SEELY 1990). Karyocytomegaly seen in this study seems to be due to direct antimitotic action of SZ, because they were detected in SZ-treated both hyperglycemic and non-hyperglycemic mice. It is a very interesting fact that many of the compounds which produce karyocytomegaly in the urinary tubule epithelial cells are also renal carcinogens (RICHARDSON and WOODARD 1986). It may be reasonable to consider that this change has some relations to adenoma or adenocarcinoma detected in the chronic phase of SZ-induced toxicity. 490
Exp Toxic Pathol 47 (1995) 6
Luminal dilatation and cyst-formation of distal tubules were observed only in hyperglycemic mice, and they seem to be secondary to persistent hyperglycemia. On the other hand, it has recently been reported that the majority of renal cell tumors of mice induced by 1,2-dimethylhydrazine arise from the distal tubules or collecting ducts (AHN et al. 1994; TURUSOV and CHEMERIS 1992). Therefore, there still remains the possibility that these changes might be an indication of renal carcinogenecity of SZ. Although it was reported that PAS-positive materials have accumulated in distal tubule epithelial cells of SZtreated rats (CHIECO et al. 1993; RASCH 1984) and DBAl2N strain of mice (HoNJO et al. 1986), this change was not observed in this study. In addition, notable changes in the basement membrane have been reported in polycystic diseases of humans and experimental animals and they are suggested as a key role in the pathogenesis of these diseases (CARONE et al. 1992, 1994; CUPPAGE et al. 1980; EBIHARA et al. 1988; KANWAR and CARONE 1984). However, any changes in the basement membrane could not be detected in this study. In conclusion, histopathological characteristics of subacute renal lesions of mice induced by SZ was clarified light and electron microscopically. In order to investigate the histogenesis of SZ-induced renal cell tumors, further histochemical studies are now in progress.
References AHN YS, CHEMERIS GY, TURUSOV VS, et al.: Enzymic pattern of preneoplastic and neoplastic lesions induced in the kidney of CBA mice by 1,2-dimethylhydrazine. Toxicol Patho11994; 22: 415-422. ARISON RN, FEUDALE EL: Induction of renal tumor by streptozotocin in rats. Nature 1967; 214: 1254-1255. CARONE FA, NAKAMURA S, CAPUTO M, et al.: Cell polarity in human renal cystic disease. Lab Invest 1994; 70: 648-655. CARONE FA, NAKAMURA S, PUNYARIT P, et al.: Sequential tubular cell and basement membrane changes in polycystic kidney disease. J Am Soc Nephrol1992; 3: 244-253. CHIECO P, VENTURINI AP, BARBANTI M, et al.: A quantitative cytochemical study on the pathogenesis of streptozotocin-induced epithelial tumors in rat kidney. Toxicol Patholl993; 21: 402-408. CORTES F, DUMLER F, GOLDMAN J, et al.: Relationship between renal function and metabolic alterations in early streptozotocin-induced diabetes in rats. Diabetes 1987; 36: 80-87. CUPPAGE FE, HUSEMAN RA, CHAPMAN A, et al.: Ultrastructure and function of cysts from human adult polycystic kidneys. Kidney Int 1980; 17: 372-38l. EBIHARA I, KILLEN PD, LAURIE GW, et al.: Altered mRNA expression of basement membrane components in a murine model of polycystic kidney disease. Lab Invest 1988; 58: 262-269. HARD GC: Identification of a high-frequency model for renal carcinoma by the induction of renal tumors in the mouse with a single dose of streptozotocin. Cancer Res 1985; 45: 703-708.
HONJO K, DOl K, DOl C, et al.: Histopathology of streptozotocin-induced diabetic DBAl2N and CD-l mice. Lab Anim 1986; 20: 298-303. HORTON L, Fox C, CORRIN B, et al.: Streptozotocin-induced renal tumors in rats. Br J Cancer 1977; 36: 692-699. Iw ASE M, KIKUCHI M, NUNOI K, et al.: A new model of type 2 (non-insulin-dependent) diabetes induced by neonatal streptozotocin treatment. Diabetologia 1986; 29: 808-818. JAKOB SON J, SIDENIUS P, GUNDERSON HJG, et al.: Quantitative changes of cerebral neocortical structure in insulintreated long-term streptozotocin-induced diabetes in rats. Diabetes 1987; 36: 597-60l. KANEDA K, IWAO J, SAKATA N, et al.: Correlation between mitochondrial enlargement in renal proximal tubules and microalbuminuria in rats with early streptozotocin-induced diabetes. Acta Pathol Jpn 1992; 42: 855-860. KANWAR YS, CARONE FA: Reversible changes of tubular cell and basement membrane in drug-induced renal cystic disease. Kidney Int 1984; 26: 35-43. KAZUMI T, YOSHINO G, FUJII S, et al.: Tumorigenic action of streptozotocin on the pancreas and kidney in male Wi star rats. Cancer Res 1978; 38: 2144-2147. KUME E, DOl C, ITAGAKI S, et al.: Glomerular lesions in unilateral nephrectomized and diabetic (UN-D) mice. J Vet Med Sci 1992; 54: 1085-1090. LIEBELT AG: Unique features of anatomy, histology, and ultrastructure, kidney, mouse. In: JONES TC, MOHR U, HUNT RD (Eds.): Urinary system. Springer-Verlag Berlin 1986; pp. 24-44.
LIKE AA, ApPEL MC, WILLIAMS RM, et al.: Streptozotocininduced pancreatic insulitis in mice. Morphologic and physiologic studies. Lab Invest 1978; 38: 470-486. MAUER SM, LEE CS, NAJARIAN JS, et al.: Induction of malignant kidney tumors in rats with streptozotocin. Cancer Res 1974;34: 158-160. MONTGOMERY CA Jr, SEELY JC: Kidney. In: BOORMAN GA, EUSTIS SL, ELWELL MR et al. (Eds.): Pathology ofthe Fischer Rat. Reference and Atlas. Academic Press San Diego 1990; pp. 127-153. OKAWA H, DOl K: Neoplastic lesions in streptozotocintreated rats. Exp Anim (Tokyo) 1983; 32: 77-84. PAYNE BJ, SAUNDERS LZ: Heavy metal nephropathy of rodents. Vet Patho11978; 15: 51-87. RAKIETEN N, GORDON BS, COONEY DA, et al.: Renal tumorigenic action of streptozotocin (NSC-85998) in rats. Cancer Chemother Rep 1968; 52: 563-567. RASCH R: Tubular lesions in streptozotocin-diabetic rats. Diabetologia 1984; 27: 32-37. RICHARDSON JA, WOODARD JC: Renal tubular karyocytomegaly, Rat. In: JONES TC, MOHR U, HUNT RD (Eds.): Urinary system. Springer-Verlag Berlin 1986; pp. 189-192. SPANHEIMER RG, UMPIERREZ GE, STUMPF V: Decreased collagen production in diabetic rats. Diabetes 1988; 37: 371-376. TURUSOV VS, CHEMERIS GY: Renal cell tumors induced in CBA male mice by 1,2-dimethylhydrazine. Toxicol Patho11992; 20: 570-575.
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Laboratory of Biomedical Science, Department of Veterinary Medical Science, Faculty of Agriculture, The University of Tokyo, Japan
Histopathology of subacute renal lesions in mice induced by streptozotocin SHIN-ICHI ITAGAKI, ETSUKO NISHIDA, MIN-JAE LEE and KUNIO DOl With 12 figures and 1 table Received: December 1, 1994; Accepted: April 25, 1995 Address for correspondence: Dr. SHIN-IeHI ITAGAKI, Laboratory of Biomedical Science, Department of Veterinary Medical Science, Faculty of Agriculture, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan. Key words: Hyperglycemia; Streptozotocin, renal lesions; Renal lesions, streptozotocin; Kidney, streptozotocin.
Summary Streptozotocin (SZ) was inoculated intraperitoneally to male and female mice of ICR and BALB/c strains in a different way of administration (A: single injection and B: consecutive 5 days-injection) and subacute renal changes were examined light and electron microscopically 8 weeks after SZ-administration. The following changes were detected: (1) reduction in the rate of male-type Bowman's capsules in male mice, (2) karyocytomegaly of proximal tubular epithelial cells, and (3) dilatation of distal tubules. Tubular changes were detected with high incidence in A (males and females) and B groups (males) ofICR strain.
Introduction Streptozotocin (SZ) has been used as a useful tool for either the induction of insulin-dependent diabetes mellitus or the investigation of its complications in laboratory rodents (CORTES et al. 1987; IWASE et al. 1986; JAKOBSON et al. 1987; LIKE et al. 1978; SPANHEIMER et al. 1988). SZ is also known as a carcinogenic agent to induce renal adenomas and carcinomas when administered to rats and mice (ARISON and FEUDALE 1967; HARD 1985; HORTON et al. 1977; KAZUMI et al. 1978; MAUER et al. 1974; OKAWA and DOl 1983; RAKIETEN et al. 1968). Although urinary tubules are affected in the subacute phase (CHIECO et al. 1993; HONJO et al. 1986; KANEDA et al. 1992; RASCH 1984), the details of the lesion and its relation to finally formed renal tumors are still unclear. In this connection, comparative bioassays conducted in rats with alloxan, a similar diabetogenic agent, suggest that neoplastic growth is due to a direct toxic effect of SZ on kidney cells and not to the induced diabetes (MAUER et al. 1974). In this study, using male and female mice of two strains and two ways of administration, we tried to esta-
blish the best experimental system for further investigations of SZ-induced renal tumors and clarify the light and electron microscopic features of SZ-induced subacute renal lesions. Pathogenesis of SZ-induced renal lesions in this study will be also discussed.
Material and methods Twenty-five each of male and female mice ofCrj:CD1 (ICR; Charles River Japan Inc., Kanagawa) and BALB/c Cr SIc strains (BALB/c; Japan SLC Inc., Shizuoka) were obtained at 7 weeks of age (initial body weights (B.W.); male ICR: 35.3 ± 2.0 g; female ICR: 26.6 ± 1.8 g; male BALB/c: 25.0 ± 1.3 g; female BALB/c: 20.5 ± 1.0 g). They were housed 5 per polycarbonate cage with wood chips for bedding under controlled conditions (temperature, 23 ± 2°C; humidity, 55 ± 5 %). Food (MF pellets, Oriental Yeast Co., Ltd., Tokyo) and water were available ad libitum. Mice of each strain were divided into 3 groups including the same number of males and females and treated intraperitoneally with SZ at 8 weeks of age as follows; A group (n = 10): single administration of 200 mg/kg B.W. of SZ; B group (n = 10): administration of 50 mg/kg B.W. SZ for 5 consecutive days; C group (n = 5): administration of citrate buffer alone for 5 consecutive days. In other words, all mice were divided into 12 groups (lCRMale (M)-A, -B and -C; ICR-Female (F)-A, -B and -C; BALB/c-M-A, -B and -C; BALB/c-F-A, -B and -C; Table 1). SZ (Sigma Chemical Company, St. Louis, MO) was dissolved in citrate buffer (pH 4.5) immediately before daily treatment. All mice were sacrificed by heart puncture under ether anesthesia 8 weeks after completion of the treatment. Kidneys were fixed in 10 % neutral buffered formalin, and 4 /lm-paraffin sections were cut and Exp Toxic Pathol 47 (1995) 6
485
stained with hematoxylin and eosin (HE). Small pieces of the kidney obtained from each mouse were fixed with 2.S % glutaraldehyde, 2.0 % paraformaldehyde in O.OS M phosphate buffer (pH 7.4), postfixed with 1 % osmium tetroxide in the same buffer and processed for electron microscopy. Ultrathin sections were double stained with uranyl acetate and lead citrate, and observed under a JEOL 1200 EX electron microscope (JEOL Co., Ltd., Tokyo). In male mice, it is well known that two types of Bowman's capsules are observed (one is lined with cuboidal to low columnar epithelia and the other with flattened epithelia). The number of each type of Bowman's capsules was counted on HE-stained coronal sections of the kidney (one section per mouse) and the ratio of Bowman's capsules lined with cuboidal to low columnar epithelia to all Bowman's capsules was calculated in all male mice. Values were expressed as mean ± SD and statistical analysis was performed using Student's t-test.
Blood glucose levels were colorimetrically measured on each serum sample using the Glucose C-test kit (Wako Pure Chemical Industries Ltd., Osaka).
Results The incidence of hyperglycemia and histopathological characteristics of renal lesions are summarized in table 1.
Blood glucose levels The average of blood glucose levels was 198.8 ± 24.96 mg/dl in C group (both sex of both strains), and animals which exhibited the blood glucose level more than 300 mg/dl were scored as hyperglycemic. All animals of ICR-M-B and ICR-F-A groups, 80 % of ICR-FB group and 60 % of ICR-M-A and BALB/c-M-A and
Table 1. Incidence of hyperglycemia and histopathological changes in the kidney. Hyperglycemia ICR-MA(10)d BOO)
+(6) -(4) +(10)
C(S)
-(S)
ICR-FAOO) BOO)
Bowman's cap." (%).
40.9 ± 14.3 e *** 71.5 ± 10.6* 24.7 ± 14.9*** 88.6 ± 6.S2
+(0) +(8) -(2)
Proximal tubules b
Distal tubules c
++(4)
++(3), +(2)
+(4)
++(2), +(7)
+(2)
++(4), +(S)
++(1), +(1)
-(5)
C(5) BALB/c-MA(10) B(10)
+(6) -(4) +(6) -(4) -(S)
C(S)
BALB/c-FA(lO) BOO)
+(1) -(9) -(10)
C(S)
-(S)
21.6 ± 8.00*** SS.S ± 17.3* 24.0 ± 7.88*** S1.5 ± 14.6** 81.8 ± 4.48
+(3) +(1)
+(1) +(1)
+(1)
+(1) +(2)
Ratio of Bowman's capsules composed of cuboidal to low columnar epithelial cells to all Bowman's capsules; Karyocytomega1y of proximal tubules (+: moderate, ++: prominent); Dilatation of distal tubules (+: mild to moderate, ++: cyst formation); d Number of parenthesis indicates number of mice; e Values are mean ± S.D. *·**·***Significantly different from C group (*p < O.OS, **p < 0.01, ***p < 0.001). a
b C
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Exp Toxic Patho1 47 (1995) 6
Fig. 1. Kidney of a mouse in ICR-M-C group. Bowman's capsule is lined with cubodal epithelium. HE. x430. Fig. 2. Kidney of a mouse in ICR-M-A group. Bowman's capsule is lined with flattened epithelium. HE. x430. Fig. 3. Kidney of a mouse in ICR-M-A group. Proximal tubule epithelial cells show moderate karyocytomegaly (arrow-
heads). HE. x430.
Fig. 4. Kidney of a mouse in ICR-M-A group. Proximal tubule epithelial cell shows prominent karyocytomegaly (arrow-
head). HE. x430.
Fig. 5. Kidney of a mouse in ICR-F-A group. Distal tubules show mild to moderate luminal dilatation. A piled-up appearance of epithelial cells (arrowhead) is seen. HE. x21S. Fig. 6. Kidney of a mouse in ICR-F-A group. Distal tubule forms a large cyst. HE. x150.
Exp Toxic PathoI 47 (1995) 6
487
Fig. 7. Cuboidal epithelial cell of Bowman's capsule of a mouse in ICR-M-A group. x4,000. Fig. 8. Flattened epithelial cell (arrowhead) of Bowman's capsule of a mouse in ICR-M-B group. x7,000. -B groups showed hyperglycemia (table 1). In contrast, only one mouse exhibited hyperglycemia in BALB/c-FA and no one in BALB/c-F group (table 1).
Light microscopic findings Light microscopic changes were mainly detected in the renal cortex. Renal corpuscle: Glomeruli of SZ-treated mice showed no prominent changes. Bowman' s capsules of male mice of C group were generally lined with cuboidal to low columar epithelia (fig. 1). On the other hand, in male mice of SZ-treated groups, the number of Bowman's capsules with cuboidal to low columnar epithelia significantly decreased and many Bowman' s capsules were lined with flattened epithelia (fig. 2). This tendency was most prominent in hyperglycemic mice of both strains (table 1). Bowman ' s capsules in female mice were lined with flattened epithelia and no differences were detected among all groups. Proximal tubule: Mild to moderate karyocytomegaly were observed in some epithelial cells in SZ-treated groups except for ICR-F-B and BALB/c-F-B groups (fig. 3). Especially in 40 % of mice of ICR-M-A group, prominet changes were detected in some epithelial cells (fig. 4). Although these features were mainly detected in hyperglycemic mice, similar changes were also observed in non-hyperglycemic BALB/C mice. 488
Exp Toxic Pathol47 ( \995) 6
Distal tubule: Mild to moderate luminal dilatation of distal tubules was observed in hyperglycemic mice of ICR-M and -F and BALB/c-F groups (fig. 5). A piled-up appearance was also seen in some distal tubule epithelia lining the dilated lumen (fig. 5). Furthermore, in some SZ-treated ICR mice, cysts which were supposed to be derived from dilated distal tubules were observed (fig. 6).
Electron microscopic findings Bowman's capsule: The ultrastructure of cuboidal to low columnar cells seen in male mice was similar to that of proximal tubule epithelial cells. Namely, they were characterized by long microvilli, prominent mitochondria, and extensive invaginations of the basolateral plasma membrane (fig. 7). Flattened epithelial cells observed in many SZ-treated male mice had poor organella and their features were similar to those of epithelial cells of Bowman's capsule in female mice (fig. 8). Proximal tubules: Epithelial cells with enlarged nuclei had electron-lucent cytoplasm and poor basal infoldings, and they lacked microvilli on the apical border (fig. 9). Aggregation of mitochondria was observed in some degenerative epithelial cells (fig. 10). Distal tubule: Epithelial cells in moderately dilated tubules had normal organella although the cytoplasm of some cells was elongated and protruded into the lumen (fig. 11). Epithelial cells of cystic tubules lacked micro-
... ..
•
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Fig. 9. Proximal tubule epithelial cell with karyocytomegaly of a mouse in ICR-M-B group. x3,400. Fig. 10. Proximal tubule epithelial cells of a mouse in ICR-M-B group. Aggregation of mitochondria (arrowhead) is seen. x3,400. Fig. 11. Epithelial cells of moderately dilated distal tubule of a mouse in ICR-M-B group. x3,400. Fig. 12. Epithelial cells of cyst-formed distal tubule of a mouse in ICR-M-B group. x3,700.
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villi and basal infoldings, and some of these epithelial cells contained many vacuoles with amorphous contents (fig. 12). Within mitochondria, electron-dense deposits were detected.
Discussion In this study, SZ was inoculated intraperitoneally to male and female mice of ICR and BALB/c strains in a different way of administration. Subacute renal changes were mainly observed in Bowman's capsules, proximal tubules and distal tubules in the cortex in some groups. Tubular changes were detected with high incidence in ICR-M-A and -B and ICR-F-A groups. Prominent karyocytomegaly in proximal tubule epithelial cells was seen especially in ICR-M-A group. These data suggest that the above-mentioned combination of strain of mice, sex and way of inoculation is considered to be best as an experimental system for further investigation of the origin of tumor cells in the kidney of SZ-treated mice. As mentioned above, Bowman's capsules of sexually mature male mice are generally lined with cuboidal to low columnar epithelia. They are called "male-type of Bowman's capsules", and their epithelial cells are suggested to be similar to those of proximal tubules functionally as well as morphologically (LIEBELT 1986). In this study, male-type of Bowman's capsule were seen in approximately 80 % of renal corpuscles in control male mice of both strains. The ratio of male-type Bowman's capsule significantly decreased by SZ-treatment and this tendency was enhanced by hyperglycemia. Diabetic conditions might influence the morphology of male-type epithelial cells probably through continuously increased hydrostatic pressure in the glomerular cavity due to marked and persistent urine production (KUME et al. 1992). However, an other agent is considered to play an additional role in the initiation of this change. Because, the ratio of male-type of Bowman's capsule was statistically lower even in non-hyperglycemic mice than in control mice. Karyocytomegaly of renal tubule epithelial cells is reported in rats following exposure to some renal toxicants and carcinogens such as aflatoxin Bland G l' ochratoxin, nitrosourea, bromodichloromethane, and some heavy metal compounds (MONTGOMERY and SEELY 1990; PAYNE and SAUNDERS 1978; RICHARDSON and WOODARD 1986). Although the pathogenesis is not known, many agents causing karyocytomegaly inhibit mitosis (MONTGOMERY and SEELY 1990). Karyocytomegaly seen in this study seems to be due to direct antimitotic action of SZ, because they were detected in SZ-treated both hyperglycemic and non-hyperglycemic mice. It is a very interesting fact that many of the compounds which produce karyocytomegaly in the urinary tubule epithelial cells are also renal carcinogens (RICHARDSON and WOODARD 1986). It may be reasonable to consider that this change has some relations to adenoma or adenocarcinoma detected in the chronic phase of SZ-induced toxicity. 490
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Luminal dilatation and cyst-formation of distal tubules were observed only in hyperglycemic mice, and they seem to be secondary to persistent hyperglycemia. On the other hand, it has recently been reported that the majority of renal cell tumors of mice induced by 1,2-dimethylhydrazine arise from the distal tubules or collecting ducts (AHN et al. 1994; TURUSOV and CHEMERIS 1992). Therefore, there still remains the possibility that these changes might be an indication of renal carcinogenecity of SZ. Although it was reported that PAS-positive materials have accumulated in distal tubule epithelial cells of SZtreated rats (CHIECO et al. 1993; RASCH 1984) and DBAl2N strain of mice (HoNJO et al. 1986), this change was not observed in this study. In addition, notable changes in the basement membrane have been reported in polycystic diseases of humans and experimental animals and they are suggested as a key role in the pathogenesis of these diseases (CARONE et al. 1992, 1994; CUPPAGE et al. 1980; EBIHARA et al. 1988; KANWAR and CARONE 1984). However, any changes in the basement membrane could not be detected in this study. In conclusion, histopathological characteristics of subacute renal lesions of mice induced by SZ was clarified light and electron microscopically. In order to investigate the histogenesis of SZ-induced renal cell tumors, further histochemical studies are now in progress.
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