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A study of growth factors in human renal cysts with or without renal cell carcinoma
Clinica Chimica Acta, 217 (1993) 199-203 © 1993 Elsevier Science Publishers B.V. All rights reserved. 0009-8981/93/$06.00
199
CCA05538
Short C o m m u n i c a t i o n
A study of growth factors in human renal cysts with or without renal cell carcinoma Masakatsu Taide, Sigeru Kanda, Jiro Eguchi, Tsukasa Igawa, Hiroshi Kanetake and Y u t a k a Saito Department of Urology. Nagasaki University School of Medicine, Nagasaki (Japan) (Received 29 September 1992; revision received 16 February 1993; accepted 20 February 1993) Key words: Renal cyst fluid; Epidermal growth factor; Insulin-like growth factor-i: Transforming growth factor-//, lnterleukin-6
Introduction
Although simple renal cyst is the most common form of renal cystic disease [1], the mechanism of occurrence or enlargement in the majority of cases remains unclear. In some cases, renal cell carcinoma occasionally arises from the cells lining the cyst wall, but its etiology is also unknown. The wall of the renal cyst is known to consist of renal tubular cells [2], leading som0~tc~postulate that extension of renal cyst is caused not only by the accumulation of cyst fluid but also by the growth of cells lining the cyst wall. We have investigated the potential role of cytokines in the growth of rabbit renal tubular cells and have found that EGF, IGF-I, fibroblast growth factor (FGF) [3], hepatocyt¢ growth factor (HGF) [4] and IL-I~ [51 stimulated the growth of tubular cells, while TGF-/~ [3] and IL-6 [51 inhibited growth. Then, to examine the relationship between growth factors in renal cyst fluid and enlargement of renal cyst fluid or complication of renal cell carcinoma, a quantitative analysis of the growth factors, including EGF, lt3~-l, TGF-~ and IL-6, in human renal cyst fluids with or without renal cell carcinoma was conducted. Materials and Methods
Collection of human renal cyst fluid Cyst fluid~ of 9 renal cell carcinoma-associated cysts of 7 patients (the malignant Correspondence to: Masakatsu Taide, Department of Urology, Nagasaki University School of Medicine, Nagasaki 852, Japan.
200
group: 7 men; mean patient age was 58.4 years, with a range of 34-73 years) and of 45 simple renal cysts of 35 patients (the benign group: 14 men and 21 women; mean patient age was 64.5 years, with a range of 20-79 years) treated in our department from December 1987 to September 1991 were examined. Cyst fluids of the malignant group were collected by needle aspiration of cysts showing renal cell carcinoma of cyst wall after radical nephrectomy and fluids of the benign group were collected by percutaneous needle aspiration. All cyst fluids were aliquoted into polypropylene tubes and were stored at -20°C until use.
Measurement of immunoreactive EGF, IGF-L insulin, IL-I~ and IL-6 Immunoreactive EGF was measured by enzyme immu, oassay i~sing rabbit antihuman EGF antibody F(ab')2 and ~t-galactosidase-rabbit anti-human EGF Fab' antibody obtained from Wakunaga Pharmaceuticals, Hiroshima, Japan. Immunoreactive IGF-I was measured using a radioimmunoassay kit, the Insulin-like Growth Factor-I/Somatomedin C Reagent Pack for RIA from Amersham Japan, Tokyo, Japan. Immunoreactive insulin was measured using the Grazime insulin-EIA test from Wako Pure Chemicals, Osaka, Japan. Immunoreactive IL-I/~ was measured using the IL-I~ assay kit from Otsuka Assay Laboratory, Tokusima, Japan. immunoreactive IL-6 was measured using the PREDICTA lnterleukin-6 kit, Genzyme Corporation, Cambridge, MA.
Assay of TGF-[3 activity TGF-B activity was assessed by bioassay including stimulation of colony formation of NRK-49F cells in soft agar and the inhibition of DNA synthesis of cultured rat hepatocytes [6]. NRK-49F cells were kindly provided by the Japanese Cancer Research Resources Bank. TGF-~ activities detected by the stimulation of the colony formation of NRK.49F cells in soft agar in the presence of EGF and the inhibi. tion of the DNA synthesis of rat hepatocytes had a close relationship; therefore, an assay of DNA synthesis of cultured rat hepatocytes was examined. In addition, this TGF./~ activity was tested for neutralization by anti-TGF-Bl neutralized antibody (purified Rabbit IgG) from R & D systems, Minneapolis, MN.
Statistical analysis All values are expressed as mean ± S.D.(range). All values were analyzed by Wilcoxon's rank test. Regression lines between growth factor and simple cyst size were fitted by Pearson's correlation coefficient.
Results Immunoreactive EGF content in cyst fluid was 7.3 -,- 12.6 pg/ml (0-54 pgtml) in the benign group compared with 18.8 ± 23.0 pg/ml (0-62 pg/ml) in the malignant group (Fig. IA). Immunoreactive IGF-I content in cyst fluid was 976.9 :J: 505 pgtml (30-2,0°i0 pgtml) in the benign group and 698.8 ± 816.5 pg/ml (0-2,240 pg/ml) in the malignant group (Fig. IB). Correlation coefficient between cyst size and IGF-I content was 0.29 in the benign group (Fig. 2A).
201 80
E
o.
......
~500
A
2000
60
Ot
1500
"
1000
#b
40
t.k.
"'
"
20
?
SO0
s O
benign (n:43) 800
malignant. (n-9)
m
malignant (n=8)
(n:30) 3000
C
D
A
.,...,
E
600
2000
400
", ooo
LL
,-
-J
200 , benign (n:44)
. . . .
0 malignant (n:8)
.... benign (n:33)
A"• malignant (n:9)
Fig. I, Growth factor contents in human renal cysts with or without renal cell carcinoma. The methods for measurement of these growth factors are described in the text. EGF, epidermal growth factor; IGF-I, insulin-like growth factor-l; TGF-/~ activity, transforming growth factor-/~ activity (TGF-/~ activity resulting in half ~he maximal inhibition of DNA synthesisof rat hepatocyteswas arbitrarily defined as one unit): !1.6, interleukin,6; n, number of cyst fluid samples. The bar shows mean value. TGF-/~ activity was not detected in untreated cyst fluid of either the benign or malignant groups. In acid-treated cyst fluid, TGF-8 activity was found to be 81.8 -,- 115,7 unit/ml (0-625 unit/ml) in the benign group and 227.5 ± 233.5 unit/ml (0-667 unit/ml) in the malignant group (Fig. IC). This TGF-/~ activity was neutralized by anti-TGF-~ antibody using DNA synthesis of cultured rat hepatocyte. Correlation coefficient between cyst size and TGF-~ activity was -0.24 in the benign group (Fig. 2B). There was no significant difference between the content of EGF, IGF-I and TGF-/~ in benign and malignant cysts. lmmunoreactive IL-6 content in renal cyst fluid was 127.9 • 206.2 pg/ml (63.2-1,241.4 pg/ml) in the benign group and 636.7 4. 959.3 pg/ml (70.1-2,511.6 pg/ml) in the malignant group (Fig. ID), with a higher concentration of IL-6 in the malignant group than in the benign group (P - 0.09). insulin and IL-18 were not detected in human renal cyst fluid with or without renal cell carcinoma. Discussion
This study demonstrated that various growth factors are present in human renal cyst tluid. Among them, EGF, which is a potent growth stimulator of renal tubular
202
1500 ~"A
n- 28 r- 0.29
•
i
E C)
4)
_s/5o
e e
0
t ; . . ~ ' ~ . . q ~•
. ..
~qD .
-
]GI~-[ (pg/ml) 1500 B
2000
n:36 r- -0.24
m
E
,,,750 E =
100
3O0 S00 TGF. ~$ ( U / ml)
Fi 8. :2. Analysis of ~urrelations between fluid volume awedgrowth factor contents in human simple renal cysts. There were no significant correlations between cyst fluid volume and IGF-! contents and TGF-~ activity, A, IGF.I; B, TGF-B.
cells, was present in very small amounts in both benign and malignant groups. EGF stimulates the growth of cultured rabbit renal t,bular cells in a dose-dependent manner from 0.2 to 10 ng/ml [31. Thus, it can be said that EGF in cyst fluid may not be able to stimulate the growth of the cells li~aing the renal cyst wall. The concentration of IOF=I in cyst fluid wa, high enough to stimulate the growth of cells lining the cyst wall [3]. It has been reported ~hat IOF-I is produced by renal collecting tubular cells [7] and that EGF is mainly produced by distal tubular cells [8]. Renal simple cyst fluid used in this study was found to contain large amounts of IGF-I and little EGF. Thus, the cells on the cyst wall may originate from the collecting tubules. It is well known that TGF=/~ is produced in a latent form and can be activated by various mechanisms, including acidification and heating [9]. In this study, we found that all TGF-/3 activity was in a latent form in cyst fluid. TGF-/3 is reported as a potent growth inhibitor for renal tubul.~r cells [3,101 and the fact that all TGF-/~ activity is present in a latent form may be viewed as convenient in that the growth of cells on the cyst wall could not be inhibited.
203
It has been reported that I£.6 acts as an autocrine growth factor for renal carcinoma cells [11]. It may follow that the renal carcinoma cells can produce IL-6, leading to a high concentration of IL-6 in some malignant cyst fluids. The assay of IL-6 in more cyst fluids may facilitate the diagnosis of renal cell carcinoma associated with renal cysts. In conclusion, a high concentration of IGF-I and the latency of TGF-/~ in renal cyst fluid may be correlated with the growth of renal cysts. Acknowledgements
We thank T. Shimogama for technical assistance and M. Yoshimoto for help. This work was supported by a Grant-in-Aid for Co-operative Resear~,h (A) (02304047) from the Ministry of Education, Science and Culture, Japan. References ! 2 3 4 $ 6 7 8 9 10 11
Bernstein J, Gardner KD Jr. Renal cystic disease and renal dysplasia. In: Walsh PK, Gittes RF, Perlmutter AD, Stamey TA, eds. Campbell's Urology. Fifth edition. Philadelphia: W.B. Saunders, 1986;1760-1803. Grantham JJ. Control of renal cyst formation and enlargement. In: Hatano M, ed. Nephrology. Vol. 2. Tokyo: Springer-Verlag, 1991;! 524-1529. Kanda S, Nomata H, Saha PK et al. Growth regulation of the renal cortical tubular cells by epidermal growth factor, insulin-like growth factor-l, acidic and basic fibroblast growth factor, and transforming growth factor-B in serum free culture. Cell Biol Int Rep 1989;13:68"7-699. lgawa T, Kanda S, Kanetake H et al. Hepatocyte growth factor is a potent mitogen for cultured rabbit renal tubular epithelial cells. Biochem Biophys Res Commun 1991;I74:831-838. Kanda S, Taide M, lgawa T et al. Effects ofcytokines on the growth ofcultured rabbit renal cortical tubular cells, Jpn J Nephrol 1992;34:19-25. Saha PK, Kanda S, Morimitsu H, Nishimura N, Kanetake H, Saito Y. Transforming growth factorO-like activity in human hydrocele fluid. Urol Res 1990;18:295-298, Hammerman MR, Growth hormone, insulin-like growth factor-i, and kidney. In: Hatano M, ed. Nephrology. Vol. 2, Tokyo: Springer-Verlag, 1991;1304-1310. Rail LB, Scott J, Bell G! et al. Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature 1985;3i 3:228-23 !. MiyazonoK, Hellman U, Wernstedt C, Heldin C-H. Latent high molecular weight complex of transforming growth factor~l. J Biol Chem 1988;263:6407-6415. Fine LG, Holley RW, Nasri H, Badei-Dezfooly B. BSC-I growth inhibitor transforms a mitogenic stimulus into a hypertrophic stimulus for renal proximal tubular cells: relationship to Na+/H÷ antiport activity. Proc Natl Acad Sci USA 1985;82:6163-6166. Miki S, lwano M, Miki Y et al. lnterleukin-6(IL-6) functions as an in vitro aut~rine growth factor in renal cell carciromas. Fed Eur Biochem Soc Lett 1989;250:607-610.
199
CCA05538
Short C o m m u n i c a t i o n
A study of growth factors in human renal cysts with or without renal cell carcinoma Masakatsu Taide, Sigeru Kanda, Jiro Eguchi, Tsukasa Igawa, Hiroshi Kanetake and Y u t a k a Saito Department of Urology. Nagasaki University School of Medicine, Nagasaki (Japan) (Received 29 September 1992; revision received 16 February 1993; accepted 20 February 1993) Key words: Renal cyst fluid; Epidermal growth factor; Insulin-like growth factor-i: Transforming growth factor-//, lnterleukin-6
Introduction
Although simple renal cyst is the most common form of renal cystic disease [1], the mechanism of occurrence or enlargement in the majority of cases remains unclear. In some cases, renal cell carcinoma occasionally arises from the cells lining the cyst wall, but its etiology is also unknown. The wall of the renal cyst is known to consist of renal tubular cells [2], leading som0~tc~postulate that extension of renal cyst is caused not only by the accumulation of cyst fluid but also by the growth of cells lining the cyst wall. We have investigated the potential role of cytokines in the growth of rabbit renal tubular cells and have found that EGF, IGF-I, fibroblast growth factor (FGF) [3], hepatocyt¢ growth factor (HGF) [4] and IL-I~ [51 stimulated the growth of tubular cells, while TGF-/~ [3] and IL-6 [51 inhibited growth. Then, to examine the relationship between growth factors in renal cyst fluid and enlargement of renal cyst fluid or complication of renal cell carcinoma, a quantitative analysis of the growth factors, including EGF, lt3~-l, TGF-~ and IL-6, in human renal cyst fluids with or without renal cell carcinoma was conducted. Materials and Methods
Collection of human renal cyst fluid Cyst fluid~ of 9 renal cell carcinoma-associated cysts of 7 patients (the malignant Correspondence to: Masakatsu Taide, Department of Urology, Nagasaki University School of Medicine, Nagasaki 852, Japan.
200
group: 7 men; mean patient age was 58.4 years, with a range of 34-73 years) and of 45 simple renal cysts of 35 patients (the benign group: 14 men and 21 women; mean patient age was 64.5 years, with a range of 20-79 years) treated in our department from December 1987 to September 1991 were examined. Cyst fluids of the malignant group were collected by needle aspiration of cysts showing renal cell carcinoma of cyst wall after radical nephrectomy and fluids of the benign group were collected by percutaneous needle aspiration. All cyst fluids were aliquoted into polypropylene tubes and were stored at -20°C until use.
Measurement of immunoreactive EGF, IGF-L insulin, IL-I~ and IL-6 Immunoreactive EGF was measured by enzyme immu, oassay i~sing rabbit antihuman EGF antibody F(ab')2 and ~t-galactosidase-rabbit anti-human EGF Fab' antibody obtained from Wakunaga Pharmaceuticals, Hiroshima, Japan. Immunoreactive IGF-I was measured using a radioimmunoassay kit, the Insulin-like Growth Factor-I/Somatomedin C Reagent Pack for RIA from Amersham Japan, Tokyo, Japan. Immunoreactive insulin was measured using the Grazime insulin-EIA test from Wako Pure Chemicals, Osaka, Japan. Immunoreactive IL-I/~ was measured using the IL-I~ assay kit from Otsuka Assay Laboratory, Tokusima, Japan. immunoreactive IL-6 was measured using the PREDICTA lnterleukin-6 kit, Genzyme Corporation, Cambridge, MA.
Assay of TGF-[3 activity TGF-B activity was assessed by bioassay including stimulation of colony formation of NRK-49F cells in soft agar and the inhibition of DNA synthesis of cultured rat hepatocytes [6]. NRK-49F cells were kindly provided by the Japanese Cancer Research Resources Bank. TGF-~ activities detected by the stimulation of the colony formation of NRK.49F cells in soft agar in the presence of EGF and the inhibi. tion of the DNA synthesis of rat hepatocytes had a close relationship; therefore, an assay of DNA synthesis of cultured rat hepatocytes was examined. In addition, this TGF./~ activity was tested for neutralization by anti-TGF-Bl neutralized antibody (purified Rabbit IgG) from R & D systems, Minneapolis, MN.
Statistical analysis All values are expressed as mean ± S.D.(range). All values were analyzed by Wilcoxon's rank test. Regression lines between growth factor and simple cyst size were fitted by Pearson's correlation coefficient.
Results Immunoreactive EGF content in cyst fluid was 7.3 -,- 12.6 pg/ml (0-54 pgtml) in the benign group compared with 18.8 ± 23.0 pg/ml (0-62 pg/ml) in the malignant group (Fig. IA). Immunoreactive IGF-I content in cyst fluid was 976.9 :J: 505 pgtml (30-2,0°i0 pgtml) in the benign group and 698.8 ± 816.5 pg/ml (0-2,240 pg/ml) in the malignant group (Fig. IB). Correlation coefficient between cyst size and IGF-I content was 0.29 in the benign group (Fig. 2A).
201 80
E
o.
......
~500
A
2000
60
Ot
1500
"
1000
#b
40
t.k.
"'
"
20
?
SO0
s O
benign (n:43) 800
malignant. (n-9)
m
malignant (n=8)
(n:30) 3000
C
D
A
.,...,
E
600
2000
400
", ooo
LL
,-
-J
200 , benign (n:44)
. . . .
0 malignant (n:8)
.... benign (n:33)
A"• malignant (n:9)
Fig. I, Growth factor contents in human renal cysts with or without renal cell carcinoma. The methods for measurement of these growth factors are described in the text. EGF, epidermal growth factor; IGF-I, insulin-like growth factor-l; TGF-/~ activity, transforming growth factor-/~ activity (TGF-/~ activity resulting in half ~he maximal inhibition of DNA synthesisof rat hepatocyteswas arbitrarily defined as one unit): !1.6, interleukin,6; n, number of cyst fluid samples. The bar shows mean value. TGF-/~ activity was not detected in untreated cyst fluid of either the benign or malignant groups. In acid-treated cyst fluid, TGF-8 activity was found to be 81.8 -,- 115,7 unit/ml (0-625 unit/ml) in the benign group and 227.5 ± 233.5 unit/ml (0-667 unit/ml) in the malignant group (Fig. IC). This TGF-/~ activity was neutralized by anti-TGF-~ antibody using DNA synthesis of cultured rat hepatocyte. Correlation coefficient between cyst size and TGF-~ activity was -0.24 in the benign group (Fig. 2B). There was no significant difference between the content of EGF, IGF-I and TGF-/~ in benign and malignant cysts. lmmunoreactive IL-6 content in renal cyst fluid was 127.9 • 206.2 pg/ml (63.2-1,241.4 pg/ml) in the benign group and 636.7 4. 959.3 pg/ml (70.1-2,511.6 pg/ml) in the malignant group (Fig. ID), with a higher concentration of IL-6 in the malignant group than in the benign group (P - 0.09). insulin and IL-18 were not detected in human renal cyst fluid with or without renal cell carcinoma. Discussion
This study demonstrated that various growth factors are present in human renal cyst tluid. Among them, EGF, which is a potent growth stimulator of renal tubular
202
1500 ~"A
n- 28 r- 0.29
•
i
E C)
4)
_s/5o
e e
0
t ; . . ~ ' ~ . . q ~•
. ..
~qD .
-
]GI~-[ (pg/ml) 1500 B
2000
n:36 r- -0.24
m
E
,,,750 E =
100
3O0 S00 TGF. ~$ ( U / ml)
Fi 8. :2. Analysis of ~urrelations between fluid volume awedgrowth factor contents in human simple renal cysts. There were no significant correlations between cyst fluid volume and IGF-! contents and TGF-~ activity, A, IGF.I; B, TGF-B.
cells, was present in very small amounts in both benign and malignant groups. EGF stimulates the growth of cultured rabbit renal t,bular cells in a dose-dependent manner from 0.2 to 10 ng/ml [31. Thus, it can be said that EGF in cyst fluid may not be able to stimulate the growth of the cells li~aing the renal cyst wall. The concentration of IOF=I in cyst fluid wa, high enough to stimulate the growth of cells lining the cyst wall [3]. It has been reported ~hat IOF-I is produced by renal collecting tubular cells [7] and that EGF is mainly produced by distal tubular cells [8]. Renal simple cyst fluid used in this study was found to contain large amounts of IGF-I and little EGF. Thus, the cells on the cyst wall may originate from the collecting tubules. It is well known that TGF=/~ is produced in a latent form and can be activated by various mechanisms, including acidification and heating [9]. In this study, we found that all TGF-/3 activity was in a latent form in cyst fluid. TGF-/3 is reported as a potent growth inhibitor for renal tubul.~r cells [3,101 and the fact that all TGF-/~ activity is present in a latent form may be viewed as convenient in that the growth of cells on the cyst wall could not be inhibited.
203
It has been reported that I£.6 acts as an autocrine growth factor for renal carcinoma cells [11]. It may follow that the renal carcinoma cells can produce IL-6, leading to a high concentration of IL-6 in some malignant cyst fluids. The assay of IL-6 in more cyst fluids may facilitate the diagnosis of renal cell carcinoma associated with renal cysts. In conclusion, a high concentration of IGF-I and the latency of TGF-/~ in renal cyst fluid may be correlated with the growth of renal cysts. Acknowledgements
We thank T. Shimogama for technical assistance and M. Yoshimoto for help. This work was supported by a Grant-in-Aid for Co-operative Resear~,h (A) (02304047) from the Ministry of Education, Science and Culture, Japan. References ! 2 3 4 $ 6 7 8 9 10 11
Bernstein J, Gardner KD Jr. Renal cystic disease and renal dysplasia. In: Walsh PK, Gittes RF, Perlmutter AD, Stamey TA, eds. Campbell's Urology. Fifth edition. Philadelphia: W.B. Saunders, 1986;1760-1803. Grantham JJ. Control of renal cyst formation and enlargement. In: Hatano M, ed. Nephrology. Vol. 2. Tokyo: Springer-Verlag, 1991;! 524-1529. Kanda S, Nomata H, Saha PK et al. Growth regulation of the renal cortical tubular cells by epidermal growth factor, insulin-like growth factor-l, acidic and basic fibroblast growth factor, and transforming growth factor-B in serum free culture. Cell Biol Int Rep 1989;13:68"7-699. lgawa T, Kanda S, Kanetake H et al. Hepatocyte growth factor is a potent mitogen for cultured rabbit renal tubular epithelial cells. Biochem Biophys Res Commun 1991;I74:831-838. Kanda S, Taide M, lgawa T et al. Effects ofcytokines on the growth ofcultured rabbit renal cortical tubular cells, Jpn J Nephrol 1992;34:19-25. Saha PK, Kanda S, Morimitsu H, Nishimura N, Kanetake H, Saito Y. Transforming growth factorO-like activity in human hydrocele fluid. Urol Res 1990;18:295-298, Hammerman MR, Growth hormone, insulin-like growth factor-i, and kidney. In: Hatano M, ed. Nephrology. Vol. 2, Tokyo: Springer-Verlag, 1991;1304-1310. Rail LB, Scott J, Bell G! et al. Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature 1985;3i 3:228-23 !. MiyazonoK, Hellman U, Wernstedt C, Heldin C-H. Latent high molecular weight complex of transforming growth factor~l. J Biol Chem 1988;263:6407-6415. Fine LG, Holley RW, Nasri H, Badei-Dezfooly B. BSC-I growth inhibitor transforms a mitogenic stimulus into a hypertrophic stimulus for renal proximal tubular cells: relationship to Na+/H÷ antiport activity. Proc Natl Acad Sci USA 1985;82:6163-6166. Miki S, lwano M, Miki Y et al. lnterleukin-6(IL-6) functions as an in vitro aut~rine growth factor in renal cell carciromas. Fed Eur Biochem Soc Lett 1989;250:607-610.