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Transforming Growth Factor-β1, Vascular Endothelial Growth Factor, and Bone Morphogenic Protein-7 Expression in Tacrolimus-Induced Nephrotoxicity in Rats
Transforming Growth Factor-1, Vascular Endothelial Growth Factor, and Bone Morphogenic Protein-7 Expression in Tacrolimus-Induced Nephrotoxicity in Rats B. Ög˘ütmen, S. Tug˘lular, F. Çakalag˘aog˘lu, Ç. Özener, and E. Akog˘lu ABSTRACT The aim of our study was to investigate transforming growth factor (TGF)-1, vascular endothelial growth factor (VEGF), and bone morphogenic protein-7 (BMP-7) expression in the rat model of chronic tacrolimus (TAC) toxicity compared to healthy controls. Seventeen male Wistar rats were divided into two groups: group 1 animals were healthy controls and Group 2 animals were treated with TAC (1 mg/kg per day intraperitoneally for 8 weeks). At the end of the study period the animals were sacrificed following renal function studies including blood urea nitrogen (BUN), serum creatinine, and creatinine clearance, and renal tissues were examined by light microscopy for the findings of tacrolimus toxicity, specifically for afferent arteriolopathy and interstitial fibrosis. TGF-1, VEGF, and BMP-7 expression were assessed by semiquantative scoring of the immunohistochemically stained specimens. Mean TAC levels were 5.53 ⫾ 2.38 ng/mL in group 2. BUN, creatinine levels, and creatinine clearance were 57.99 ⫾ 11.13 vs 39.49 ⫾ 5.64 mg/dL; 0.60 ⫾ 0.16 vs 0.65 ⫾ 0.09 mg/dL; 0.97 ⫾ 0.39 vs 1.17 ⫾ 0.32 mL/min in group 2 versus group 1. Only the BUN level was significantly higher in group 2 compared to group 1. Afferent arteriolopathy and interstitial fibrosis were significantly increased in group 2 compared to group 1. TGF-1 and VEGF expression was significantly increased while BMP-7 expression was significantly decreased in group 2 versus group 1. In conclusion, our findings suggest that TAC-induced nephrotoxicity is associated with increased TGF-1 and VEGF and decreased BMP-7 expression.
T
ACROLIMUS (TAC) is a calcineurin inhibitor used for the prevention of acute rejection following solid organ transplantation. Chronic nephrotoxicity limits its long term use.1–3 Chronic TAC nephrotoxicity is characterized by renal functional impairment preceded by some welldefined histopathological changes including arteriolar hyalinosis, tubular vacuolization and calcification, and striped interstitial fibrosis. Several growth factors and cytokines has been suggested to be responsible for the occurrence of these morphological findings. Vascular endothelial growth factor (VEGF),4 which is secreted by epithelial cells, myocytes, and macrophages, stimulates endothelial cells mitogenesis and increases their fenestrations thus enhancing permeability.5,6 Another member of the transforming growth factor (TGF)- superfamily is the bone morphogenic protein (BMP)-7, which has been reported to prevent the progression of renal fibrosis in several animal models7,8 via counteracting some profibrogenic actions of TGF-1. © 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 38, 487– 489 (2006)
In this study we sought to explore the expression of VEGF, TGF-1, and BMP-7 in a rat model of chronic TAC nephrotoxicity. MATERIALS AND METHODS The study included 17 Wistar male rats of 2 to 4 months old weighing 220 to 310g. They were kept as quartet groups in cages under controlled heat, humidity, and light conditions and fed a salt-free diet with ad lib access to water. Study animals were randomized to two groups. Group 1, the control group (n ⫽ 8), received 1 mL/kg per day distilled water intraperitoneally (ip), From the Department of Pathology, Division of Nephrology, Marmara University Medical School, Istanbul, Turkey. This study was supported by Ezcacibas¸i and tacrolimus amp was provided by Fujisawa, Japan. Address reprint requests to Dr Betül Ög˘ütmen, Gardenya 4/1 D 3 62 Ada Atas¸ehir, Istanbul, Turkey. E-mail: betulogutmen@ yahoo.com 0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2005.12.048 487
ÖG˘U¨TMEN, TUG˘LULAR, ÇAKALAG˘AOG˘LU ET AL
488 while group 2, the TAC group (n ⫽ 9), received TAC 1 mg/kg/d ip for 30 days. The one extra animal was sacrificed at the end of the study period to confirm the development of chronic nephrotoxicity. At the end of the study period, the animals were put in metabolic cages for 24-hour urine collections. The animals were then sacrificed following deep ether anesthesia with intracardiac deprivation of 10 to 12 mL blood samples for the measurement of glucose, urea, sodium, potassium, and tacrolimus levels. Via a midabdominal incision kidneys were removed for morphological examination after fixation in a 10% formol solution. Renal tissues were examined for evidence of TAC toxicity including tubulointerstitial damage and arteriolar hyalinosis. The degree of tubulointerstitial fibrosis was evaluated semiquantitatively in trichromestained specimens. Hyalinosis of the afferent arterioles was determined by counting the juxtaglomerular arterioles available for examination with a minimum of 50 glomeruli per PAS-stained specimen. They were scored semiquantitatively using immunohistochemical reagents for TGF-1 (NCL-TGFB; Novocastra), VEGF (C-1; sc-7269; Santa Cruz Biotechnology), and BMP-7 (N-19; sc-6899; Santa Cruz Biotechnology). Histological analyses were performed by two independent pathologists blinded to each other and to the groups. Urine and plasma creatinine, plasma glucose, sodium, potassium, and urea measurements were performed by spectrophotometric methods using an 917 autoanalyzer. Creatinine clearance (mL/dk) was calculated with the usual formula. Blood TAC level was measured by the IMX MCIA microparticle enzyme immunoassay method using tacrolimus II reagent pac Ref 3CID-20 commercial kit. Statistical analyses were performed with the GraphPad Prizma V.3 commercially available computer program. Descriptive statistics included Mann-Whitney U test for continuous variables and chi-square test for quantitative variables. Correlation between the variables was determined by the Spearman correlation test. Statistical significance was assumed when P ⬍ .05.
RESULTS
At the end of the study period, the weight of the animals was significantly lower in group 2 compared to group 1 (273.67 ⫾ 21.92 vs 347.50 ⫾ 50.70; P ⬍ .01). The mean TAC level of group 2 animals was 5.53 ⫾ 2.38. The other biochemical results are shown in Table 1. Histopathological and immunohistochemical findings of TAC nephrotoxicity are presented in tables 2 and 3, respectively. When fibrosis intensity and BMP-7, VEGF, and TGF-1 intensity were compared, there was a statistically positive correlation between fibrosis intensity and VEGF Table 1. Biochemical Findings of the Two Groups
Tacrolimus Glucose BUN Creatinine Na K Urine creatinine Urine volume Creatinine clearance
TAC group (n ⫽ 9)
Control group (n ⫽ 8)
P
5.53 ⫾ 2.38 235.56 ⫾ 90.81 57.99 ⫾ 11.13 0.60 ⫾ 0.16 138.89 ⫾ 1.17 4.16 ⫾ 0.49 67.02 ⫾ 33.65 13.22 ⫾ 4.24 0.97 ⫾ 0.39
0.10 ⫾ 0.00 137.50 ⫾ 29.11 39.49 ⫾ 5.64 0.65 ⫾ 0.09 139.75 ⫾ 2.25 4.51 ⫾ 0.31 65.81 ⫾ 30.97 16.50 ⫾ 7.19 1.17 ⫾ 0.32
⬍.0001 ⬍.01 ⬍.001 ⬎.05 ⬎.05 ⬎.05 ⬎.05 ⬎.05 ⬎.05
Table 2. Light Microscopic Histopathological Findings
Afferent arteriolopathy None (%) Early (%) Late (%) Fibrosis ⬍5 (%) 25–50 (%) ⬎50 (%)
TAC-G
Control-G
— 55.6 44.4
100 — —
— 55.6 44.4
100 — —
P ⬍ .0001.
(P ⬍ .0001) and TGF1 intensity of immunoexpression (P ⬍ .05), while there was a statistically negative correlation between fibrosis intensity and BMP-7 immunoexpression (P ⬍ .05). DISCUSSION
We investigated the functional and morphological findings of TAC nephrotoxicity as well as TGF-1, VEGF, and BMP-7 immune expression in the glomeruli and interstitium in a rat model of TAC nephrotoxicity. Histopathological examination of the renal tissues revealed the characteristic findings of TAC nephrotoxicity, such as interstitial mononuclear cell infiltration, interstitial edema, tubular irregularity, focal vacuolization, calcification, development of tubulitis, and moderate to significant hyalinization of the afferent arterioli. These findings suggest that structural impairment precedes and is not always translated into overt functional impairment. TGF1, which is a strong fibrogenic cytokine, played the most effective role in the development of TAC nephrotoxicity. VEGF is another profibrogenic cytokine while BMP-7, although a member of TGF- family, counteracts the profibrinogenic effects of TGF-1, thus interfering with the development of fibrosis.4 – 8 The roles of TGF-1 and VEGF in calcineurin toxicity have been previously shown in cyclosporine toxicity models.4 BMP-7 has also been previously reported to be downregulated in cyclosporine nephrotoxicity.9 Our results suggest that up-regulation of TGF-1 and VEGF and down-regulation of BMP-7 have important roles in the development of interstitial fibrosis due to TAC nephrotoxicity. This finding may have future implications for the therapeutic use of BMP-7 to avoid nephrotoxicity induced by calcineurin inhibitors, the currently indispensable immunosuppressives in solid organ transplantation. Table 3. TGF-1, VEGF, and BMP-7 and Immunoexpressions TAC group
TGF-1* (%) VEGF** (%) BMP-7* (%)
Control group
Mild
Moderate
High
None
Mild
44 — 55.6
33 66.7 44.4
22.2 33.3 —
37.5
62.5 75
*P ⬍ .05; **P ⬍ .01.
Moderate
High
25 75
— 25
TGF-1, VEGF, AND BMP-7 EXPRESSION
In conclusion, our results suggest that TGF-1 and VEGF may have important roles in TAC-induced nephrotoxicity, while BMP-7 exerts protective effects.
REFERENCES 1. Mayer AD, Dmitrewski J, Squifflet J-P, et al: Multicenter randomized trial comparing tacrolimus (FK506) and cyclosporine in the prevention of renal allograft rejection: a report of the European Tacrolimus Multicenter Renal Study Group. Transplantation 64:436, 1997 2. Busuttil RW, Klintmalm GB, Lake JR, et al: General guidelines for the use of tacrolimus in adult liver transplant patient [letter]. Transplantation 15:845, 1996 3. Henry ML: Cyclosporine and tacrolimus (FK506): a comparison of efficacy and safety profiles: Clin Transpl 13:209, 1999
489 4. Shihab FS, Bennet WM, Isaac J, et al: Angiotensin II regulation of vascular endothelial growth factor and receptors Flt-1 and KDR/Flk-1 in cyclosporine nephrotoxicity. Kidney Int 62:422, 2002 5. Terman BI, Dougher vermazen M, Carrion ME, et al: Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun 187:1579, 1992 6. Esser S, Wolburg K, Wolburg H, et al: Vascular endothelial growth factor induces endothelial fenestrations in vivo. J Cell Biol 140:947, 1998 7. Hruska K, Guo G, Wozniak M, et al: Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction: Am J Physiol 279:130, 2000 8. Wang S, Hirschberg R: Loss of renal tubular BMP7 during the evolution of experimental diabetic nephropathy. J Am Soc Nephrol 11:655, 2000 9. Tuglular S, Gogas Yavuz D, Çakalag˘aog˘lu F, et al: Cyclosporin-A induced nephrotoxicity is associated with decreased renal bone morphogenetic protein-7 expression in rats. Transplant Proc 36:131, 2004
T
ACROLIMUS (TAC) is a calcineurin inhibitor used for the prevention of acute rejection following solid organ transplantation. Chronic nephrotoxicity limits its long term use.1–3 Chronic TAC nephrotoxicity is characterized by renal functional impairment preceded by some welldefined histopathological changes including arteriolar hyalinosis, tubular vacuolization and calcification, and striped interstitial fibrosis. Several growth factors and cytokines has been suggested to be responsible for the occurrence of these morphological findings. Vascular endothelial growth factor (VEGF),4 which is secreted by epithelial cells, myocytes, and macrophages, stimulates endothelial cells mitogenesis and increases their fenestrations thus enhancing permeability.5,6 Another member of the transforming growth factor (TGF)- superfamily is the bone morphogenic protein (BMP)-7, which has been reported to prevent the progression of renal fibrosis in several animal models7,8 via counteracting some profibrogenic actions of TGF-1. © 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 38, 487– 489 (2006)
In this study we sought to explore the expression of VEGF, TGF-1, and BMP-7 in a rat model of chronic TAC nephrotoxicity. MATERIALS AND METHODS The study included 17 Wistar male rats of 2 to 4 months old weighing 220 to 310g. They were kept as quartet groups in cages under controlled heat, humidity, and light conditions and fed a salt-free diet with ad lib access to water. Study animals were randomized to two groups. Group 1, the control group (n ⫽ 8), received 1 mL/kg per day distilled water intraperitoneally (ip), From the Department of Pathology, Division of Nephrology, Marmara University Medical School, Istanbul, Turkey. This study was supported by Ezcacibas¸i and tacrolimus amp was provided by Fujisawa, Japan. Address reprint requests to Dr Betül Ög˘ütmen, Gardenya 4/1 D 3 62 Ada Atas¸ehir, Istanbul, Turkey. E-mail: betulogutmen@ yahoo.com 0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2005.12.048 487
ÖG˘U¨TMEN, TUG˘LULAR, ÇAKALAG˘AOG˘LU ET AL
488 while group 2, the TAC group (n ⫽ 9), received TAC 1 mg/kg/d ip for 30 days. The one extra animal was sacrificed at the end of the study period to confirm the development of chronic nephrotoxicity. At the end of the study period, the animals were put in metabolic cages for 24-hour urine collections. The animals were then sacrificed following deep ether anesthesia with intracardiac deprivation of 10 to 12 mL blood samples for the measurement of glucose, urea, sodium, potassium, and tacrolimus levels. Via a midabdominal incision kidneys were removed for morphological examination after fixation in a 10% formol solution. Renal tissues were examined for evidence of TAC toxicity including tubulointerstitial damage and arteriolar hyalinosis. The degree of tubulointerstitial fibrosis was evaluated semiquantitatively in trichromestained specimens. Hyalinosis of the afferent arterioles was determined by counting the juxtaglomerular arterioles available for examination with a minimum of 50 glomeruli per PAS-stained specimen. They were scored semiquantitatively using immunohistochemical reagents for TGF-1 (NCL-TGFB; Novocastra), VEGF (C-1; sc-7269; Santa Cruz Biotechnology), and BMP-7 (N-19; sc-6899; Santa Cruz Biotechnology). Histological analyses were performed by two independent pathologists blinded to each other and to the groups. Urine and plasma creatinine, plasma glucose, sodium, potassium, and urea measurements were performed by spectrophotometric methods using an 917 autoanalyzer. Creatinine clearance (mL/dk) was calculated with the usual formula. Blood TAC level was measured by the IMX MCIA microparticle enzyme immunoassay method using tacrolimus II reagent pac Ref 3CID-20 commercial kit. Statistical analyses were performed with the GraphPad Prizma V.3 commercially available computer program. Descriptive statistics included Mann-Whitney U test for continuous variables and chi-square test for quantitative variables. Correlation between the variables was determined by the Spearman correlation test. Statistical significance was assumed when P ⬍ .05.
RESULTS
At the end of the study period, the weight of the animals was significantly lower in group 2 compared to group 1 (273.67 ⫾ 21.92 vs 347.50 ⫾ 50.70; P ⬍ .01). The mean TAC level of group 2 animals was 5.53 ⫾ 2.38. The other biochemical results are shown in Table 1. Histopathological and immunohistochemical findings of TAC nephrotoxicity are presented in tables 2 and 3, respectively. When fibrosis intensity and BMP-7, VEGF, and TGF-1 intensity were compared, there was a statistically positive correlation between fibrosis intensity and VEGF Table 1. Biochemical Findings of the Two Groups
Tacrolimus Glucose BUN Creatinine Na K Urine creatinine Urine volume Creatinine clearance
TAC group (n ⫽ 9)
Control group (n ⫽ 8)
P
5.53 ⫾ 2.38 235.56 ⫾ 90.81 57.99 ⫾ 11.13 0.60 ⫾ 0.16 138.89 ⫾ 1.17 4.16 ⫾ 0.49 67.02 ⫾ 33.65 13.22 ⫾ 4.24 0.97 ⫾ 0.39
0.10 ⫾ 0.00 137.50 ⫾ 29.11 39.49 ⫾ 5.64 0.65 ⫾ 0.09 139.75 ⫾ 2.25 4.51 ⫾ 0.31 65.81 ⫾ 30.97 16.50 ⫾ 7.19 1.17 ⫾ 0.32
⬍.0001 ⬍.01 ⬍.001 ⬎.05 ⬎.05 ⬎.05 ⬎.05 ⬎.05 ⬎.05
Table 2. Light Microscopic Histopathological Findings
Afferent arteriolopathy None (%) Early (%) Late (%) Fibrosis ⬍5 (%) 25–50 (%) ⬎50 (%)
TAC-G
Control-G
— 55.6 44.4
100 — —
— 55.6 44.4
100 — —
P ⬍ .0001.
(P ⬍ .0001) and TGF1 intensity of immunoexpression (P ⬍ .05), while there was a statistically negative correlation between fibrosis intensity and BMP-7 immunoexpression (P ⬍ .05). DISCUSSION
We investigated the functional and morphological findings of TAC nephrotoxicity as well as TGF-1, VEGF, and BMP-7 immune expression in the glomeruli and interstitium in a rat model of TAC nephrotoxicity. Histopathological examination of the renal tissues revealed the characteristic findings of TAC nephrotoxicity, such as interstitial mononuclear cell infiltration, interstitial edema, tubular irregularity, focal vacuolization, calcification, development of tubulitis, and moderate to significant hyalinization of the afferent arterioli. These findings suggest that structural impairment precedes and is not always translated into overt functional impairment. TGF1, which is a strong fibrogenic cytokine, played the most effective role in the development of TAC nephrotoxicity. VEGF is another profibrogenic cytokine while BMP-7, although a member of TGF- family, counteracts the profibrinogenic effects of TGF-1, thus interfering with the development of fibrosis.4 – 8 The roles of TGF-1 and VEGF in calcineurin toxicity have been previously shown in cyclosporine toxicity models.4 BMP-7 has also been previously reported to be downregulated in cyclosporine nephrotoxicity.9 Our results suggest that up-regulation of TGF-1 and VEGF and down-regulation of BMP-7 have important roles in the development of interstitial fibrosis due to TAC nephrotoxicity. This finding may have future implications for the therapeutic use of BMP-7 to avoid nephrotoxicity induced by calcineurin inhibitors, the currently indispensable immunosuppressives in solid organ transplantation. Table 3. TGF-1, VEGF, and BMP-7 and Immunoexpressions TAC group
TGF-1* (%) VEGF** (%) BMP-7* (%)
Control group
Mild
Moderate
High
None
Mild
44 — 55.6
33 66.7 44.4
22.2 33.3 —
37.5
62.5 75
*P ⬍ .05; **P ⬍ .01.
Moderate
High
25 75
— 25
TGF-1, VEGF, AND BMP-7 EXPRESSION
In conclusion, our results suggest that TGF-1 and VEGF may have important roles in TAC-induced nephrotoxicity, while BMP-7 exerts protective effects.
REFERENCES 1. Mayer AD, Dmitrewski J, Squifflet J-P, et al: Multicenter randomized trial comparing tacrolimus (FK506) and cyclosporine in the prevention of renal allograft rejection: a report of the European Tacrolimus Multicenter Renal Study Group. Transplantation 64:436, 1997 2. Busuttil RW, Klintmalm GB, Lake JR, et al: General guidelines for the use of tacrolimus in adult liver transplant patient [letter]. Transplantation 15:845, 1996 3. Henry ML: Cyclosporine and tacrolimus (FK506): a comparison of efficacy and safety profiles: Clin Transpl 13:209, 1999
489 4. Shihab FS, Bennet WM, Isaac J, et al: Angiotensin II regulation of vascular endothelial growth factor and receptors Flt-1 and KDR/Flk-1 in cyclosporine nephrotoxicity. Kidney Int 62:422, 2002 5. Terman BI, Dougher vermazen M, Carrion ME, et al: Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun 187:1579, 1992 6. Esser S, Wolburg K, Wolburg H, et al: Vascular endothelial growth factor induces endothelial fenestrations in vivo. J Cell Biol 140:947, 1998 7. Hruska K, Guo G, Wozniak M, et al: Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction: Am J Physiol 279:130, 2000 8. Wang S, Hirschberg R: Loss of renal tubular BMP7 during the evolution of experimental diabetic nephropathy. J Am Soc Nephrol 11:655, 2000 9. Tuglular S, Gogas Yavuz D, Çakalag˘aog˘lu F, et al: Cyclosporin-A induced nephrotoxicity is associated with decreased renal bone morphogenetic protein-7 expression in rats. Transplant Proc 36:131, 2004