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Study of hepatocyte growth factor in cyclosporine-induced nephropathy
Study of Hepatocyte Growth Factor in Cyclosporineinduced Nephropathy S. Kasai, R. Yoshimura,
K. Sugimura,
A. Ohyama,
C
YCLOSPORINE (CsA) has been widely used for the clinical transplantation of various organs such as kidney, liver, heart, and pancreas, resulting in great improvement of graft survival due to its superior anti-rejection activity. However, this immunosuppressive drug has several side effects, the most serious being nephrotoxicity. CsAinduced nephropathy is diagnosed by some clinical data, BUN, urine volume, and so on. Recently, FK-506 has been reported to have much higher immunosuppressive activity than CsA, but with a lower incidence of nephropathy. However, if we can detect drug-induced nephropathy before the changes occur in clinical data, treating nephropathy will be easier. To address this problem, we have examined the effects of CsA and FK-506 treatment on hepatocyte growth function (HGF) in rats models. MATERIALS
AND
METHODS
Male LEW rats weighting from 200-250 g were used. Groups A (n = 3) received 0.1 mL of olive oil alone daily by intermuscular (IM) administration for 10 days. Group B (n = 3) received 30 mgikgid of CsA dissolved in 0.1 mL of olive oil IM for 10 days. Group C (n = 3) received 3 mgikgiday of FK-506 dissolved in 0.1 mL of olive oil IM for 15 days. Blood samples were drawn from the tail vein of the rats on days 0 (control), 3, 5, 7, and 10 in groups A and B, and on days 0, 3, 5, 7,9, 11, and 15 in group C. Blood samples were also collected in the tube with EDTA. HGF lcvcls were measured by ELISA.’ Rabbit anti-rat HGF antibodies were coated on a 96.well plate at 37°C for 15 h. After blocking with bovine serum albumin solution, conditioned medium was added to each well and the preparation was incubated for 2 h at 37°C. Wells were washed three times with PBS-Tween (PBS containing 0.025% Tween 20). Biotinylated polyclonal anti-rat HGF IgG was added and incubation was allowed to proceed for 2 h at 37°C. Wells were washed three times with PBS-tween, then incubated with horseradish peroxdase-conjugated streptoavidinbiotin complex in PBS-Tween. The enzyme reaction was initiated by adding substance solution composed of 50 mmol/L citric acid, 100 mmol/L sodium phosphate, 2.5 mg o-phenylenediamine per mL, and 0.015% H,O?. The enzyme reaction was halted by adding 1 mol/L H,SO,. Absorbance at 490 nm was measured. RESULTS
In the control rats, BUN and S-Cr levels were 15.5 mgidL and 0.5 mg/dL, respectively. No nephropathy was observed with the rats given olive oil alone, no elevation of BUN and
K. Harimoto,
T. Kishimoto,
and N. Yoshimura
S-Cr were observed after treatment until day 10. In addition, there was no difference in the HGF levels between the control rats and group A. The level of HGF in the untreated control rat was 0.301 ? 0.038 ng/mL. The levels of HGF in group A were 0.320 and 0.328 ngimL on the 7th and 10th days, respectively. On the other hand, in group B, after treatment with CsA, BUN was elevated to 18.6, 31.0 and 58.8 mg/dL, and S-Cr was elevated to 0.55, 0.65 and 2.1 mg/dL on the 5th, 7th and 10th days, respectively. Moreover, after treatment with CsA, the level of HGF was elevated to 0.508 ng/mL and 0.857 ng/mL on the 5th and 7th days, but was returning to a normal range on the 10th day (0.316 ng/mL). BUN and S-Cr levels started to be elevated on day 7. However, the level of HGF was elevated on day 5 in group B, two days ealier than the elevation of BUN and S-Cr levels. In group C, no elevation of BUN and S-Cr levels were observed after treatment with FK-506 until day 15. However, the HGF level increased on the 11 th and 15th days (0.444, 0.783 @mL). The HGF level in group C increased 6 days later than that in group B.
DISCUSSION
Hepatocyte Growth Factor, originally identified as a potent mitogen for mature hepatocytes,‘,’ is a stromal-derived polypeptied that targets a wide variety of cells, predominantly epithelial and endothelial cells. Unilateral nephrectomy in humans and experimental animals induces compensatory renal enlargement, a wellknown phenomenon in renal regeneration. This enlargement of the kidney must be accompanied by compensatory renal cell proliferation, and occurs predominantly in renal tubular epithelial cells. The existence of a blood-borne renotrophic factor has long been suggested, but identification has not been successful.
From the Department of Urology, Osaka City University School of Medicine (SK., R.Y., KS., A.O., K.H., T.K.) and The 2nd Department of Surgery, Kyoto Prefectural University of Medicine (N.Y.). Address reprint requests to Dr. S. Kasai, Department of Urology, Osaka City University School of Medicine, l-5-7 Asahimachi, Abeno-ku, Osaka 545, Japan.
0041-l 315/97/$17.00 PII SO041 -1345(97)00031-6
0 1997 by Elsevier Science inc. 655 Avenue of the Americas, New York, NY 10010
1724
Transplantation
Proceedings,
29, 1724-l 725 (1997)
HGF IN NEPHROPATHY
The renotrophic function of HGF was confirmed by an experiment in vivo.4,5 When recombinant human HGF was intravenously injected into mice with renal injuries caused by administration of H&l, or by the widely used antitumor drug cisplatin, HGF strongly suppressed the onset of severe renal dysfunction.5 Moreover, exogeneous HGF remarkably stimulated mitogenesis of renal tubular cells and induced rapid reconstruction of normal renal tissue structure after acute renal failure and after unilateral nephrectomy. Our data showed that the level of HGF was elevated earlier than BUN and S-Cr levels in CsA-induced nephropathy. Moreover, the results presented herein might contrib-
ute to the diagnosis of CsA-induced than by another clinical data.
nephropathy
earlier
REFERENCES
1. Yamada A, Matsumoto K, Iwanari H, et al: Biomed Res 16:105, 1995 2. Nakamura T, Nawa K, Ichihara A: Biochem Biophys Res Commun 122:1450, 1984 3. Russell E, McGowan A, Bucher R: J Cell Physiol 119:183, 1984 4. Igawa T, Matsumoto K, Kanda S, et al: Am J Physiol 265:61, 1993 5. Kawaida K, Matsumoto K, Shimazu H, et al: Proc Nat1 Acad Sci USA (in press)
K. Sugimura,
A. Ohyama,
C
YCLOSPORINE (CsA) has been widely used for the clinical transplantation of various organs such as kidney, liver, heart, and pancreas, resulting in great improvement of graft survival due to its superior anti-rejection activity. However, this immunosuppressive drug has several side effects, the most serious being nephrotoxicity. CsAinduced nephropathy is diagnosed by some clinical data, BUN, urine volume, and so on. Recently, FK-506 has been reported to have much higher immunosuppressive activity than CsA, but with a lower incidence of nephropathy. However, if we can detect drug-induced nephropathy before the changes occur in clinical data, treating nephropathy will be easier. To address this problem, we have examined the effects of CsA and FK-506 treatment on hepatocyte growth function (HGF) in rats models. MATERIALS
AND
METHODS
Male LEW rats weighting from 200-250 g were used. Groups A (n = 3) received 0.1 mL of olive oil alone daily by intermuscular (IM) administration for 10 days. Group B (n = 3) received 30 mgikgid of CsA dissolved in 0.1 mL of olive oil IM for 10 days. Group C (n = 3) received 3 mgikgiday of FK-506 dissolved in 0.1 mL of olive oil IM for 15 days. Blood samples were drawn from the tail vein of the rats on days 0 (control), 3, 5, 7, and 10 in groups A and B, and on days 0, 3, 5, 7,9, 11, and 15 in group C. Blood samples were also collected in the tube with EDTA. HGF lcvcls were measured by ELISA.’ Rabbit anti-rat HGF antibodies were coated on a 96.well plate at 37°C for 15 h. After blocking with bovine serum albumin solution, conditioned medium was added to each well and the preparation was incubated for 2 h at 37°C. Wells were washed three times with PBS-Tween (PBS containing 0.025% Tween 20). Biotinylated polyclonal anti-rat HGF IgG was added and incubation was allowed to proceed for 2 h at 37°C. Wells were washed three times with PBS-tween, then incubated with horseradish peroxdase-conjugated streptoavidinbiotin complex in PBS-Tween. The enzyme reaction was initiated by adding substance solution composed of 50 mmol/L citric acid, 100 mmol/L sodium phosphate, 2.5 mg o-phenylenediamine per mL, and 0.015% H,O?. The enzyme reaction was halted by adding 1 mol/L H,SO,. Absorbance at 490 nm was measured. RESULTS
In the control rats, BUN and S-Cr levels were 15.5 mgidL and 0.5 mg/dL, respectively. No nephropathy was observed with the rats given olive oil alone, no elevation of BUN and
K. Harimoto,
T. Kishimoto,
and N. Yoshimura
S-Cr were observed after treatment until day 10. In addition, there was no difference in the HGF levels between the control rats and group A. The level of HGF in the untreated control rat was 0.301 ? 0.038 ng/mL. The levels of HGF in group A were 0.320 and 0.328 ngimL on the 7th and 10th days, respectively. On the other hand, in group B, after treatment with CsA, BUN was elevated to 18.6, 31.0 and 58.8 mg/dL, and S-Cr was elevated to 0.55, 0.65 and 2.1 mg/dL on the 5th, 7th and 10th days, respectively. Moreover, after treatment with CsA, the level of HGF was elevated to 0.508 ng/mL and 0.857 ng/mL on the 5th and 7th days, but was returning to a normal range on the 10th day (0.316 ng/mL). BUN and S-Cr levels started to be elevated on day 7. However, the level of HGF was elevated on day 5 in group B, two days ealier than the elevation of BUN and S-Cr levels. In group C, no elevation of BUN and S-Cr levels were observed after treatment with FK-506 until day 15. However, the HGF level increased on the 11 th and 15th days (0.444, 0.783 @mL). The HGF level in group C increased 6 days later than that in group B.
DISCUSSION
Hepatocyte Growth Factor, originally identified as a potent mitogen for mature hepatocytes,‘,’ is a stromal-derived polypeptied that targets a wide variety of cells, predominantly epithelial and endothelial cells. Unilateral nephrectomy in humans and experimental animals induces compensatory renal enlargement, a wellknown phenomenon in renal regeneration. This enlargement of the kidney must be accompanied by compensatory renal cell proliferation, and occurs predominantly in renal tubular epithelial cells. The existence of a blood-borne renotrophic factor has long been suggested, but identification has not been successful.
From the Department of Urology, Osaka City University School of Medicine (SK., R.Y., KS., A.O., K.H., T.K.) and The 2nd Department of Surgery, Kyoto Prefectural University of Medicine (N.Y.). Address reprint requests to Dr. S. Kasai, Department of Urology, Osaka City University School of Medicine, l-5-7 Asahimachi, Abeno-ku, Osaka 545, Japan.
0041-l 315/97/$17.00 PII SO041 -1345(97)00031-6
0 1997 by Elsevier Science inc. 655 Avenue of the Americas, New York, NY 10010
1724
Transplantation
Proceedings,
29, 1724-l 725 (1997)
HGF IN NEPHROPATHY
The renotrophic function of HGF was confirmed by an experiment in vivo.4,5 When recombinant human HGF was intravenously injected into mice with renal injuries caused by administration of H&l, or by the widely used antitumor drug cisplatin, HGF strongly suppressed the onset of severe renal dysfunction.5 Moreover, exogeneous HGF remarkably stimulated mitogenesis of renal tubular cells and induced rapid reconstruction of normal renal tissue structure after acute renal failure and after unilateral nephrectomy. Our data showed that the level of HGF was elevated earlier than BUN and S-Cr levels in CsA-induced nephropathy. Moreover, the results presented herein might contrib-
ute to the diagnosis of CsA-induced than by another clinical data.
nephropathy
earlier
REFERENCES
1. Yamada A, Matsumoto K, Iwanari H, et al: Biomed Res 16:105, 1995 2. Nakamura T, Nawa K, Ichihara A: Biochem Biophys Res Commun 122:1450, 1984 3. Russell E, McGowan A, Bucher R: J Cell Physiol 119:183, 1984 4. Igawa T, Matsumoto K, Kanda S, et al: Am J Physiol 265:61, 1993 5. Kawaida K, Matsumoto K, Shimazu H, et al: Proc Nat1 Acad Sci USA (in press)