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A new model of chronic cyclosporine nephrotoxicity for comparative morphologic and histologic studies
A New Model of Chronic Cyclosporine Nephrotoxicity for Comparative Morphologic and Histologic Studies A. Ahmed, W. Ismail, J. Shortland, M. Karim, A. Raftery, A.M. El Nahas, and J. Haylor
S
INCE ITS introduction in 1976, cyclosporine (CsA) has markedly improved the survival of solid organ transplants and has also been beneficial for the treatment of autoimmune diseases. However, the benefits of CsA therapy have been potentially offset by the occurrence of nephrotoxicity. CsA nephrotoxicity which has been reported since the early 1980s, occurs even when low doses of the drug are used in the treatment of autoimmune disease.1 In the setting of cardiac transplantation, CsA nephrotoxicity has been related to the development of end-stage renal failure in about 10% of patients.2 The contribution of CsA nephrotoxicity to chronic allograft dysfunction is hard to determine due to many confounding etiologic factors, including ischemia/reperfusion injury, chronic rejection, and de novo nephropathies.3 Chronic renal injury induced by CsA is characterized by irreversible striped interstitial fibrosis and afferent arteriolar hyalinosis.3 The mechanisms of CsA-induced renal fibrosis are not well understood; they have been attributed to sustained afferent arteriolar vasoconstriction and arteriopathy, leading to preglomerular ischemia.4 The lesion of chronic CsA nephropathy has been difficult to reproduce in animals. Thus, the pathogenesis of this type of CsA-induced renal injury has been difficult to study.5 An animal model of chronic CsA nephrotoxicity has been described using salt depletion as stimulus to accelerate the long-term effects of CsA treatment.6 In the present study, the long-term effects of CsA treatment were potentiated by a stimulus of 30 minutes of warm ischemia (RWI). The aim of this comparative histologic and morphologic study was to introduce a model of CsA nephrotoxicity without using either higher doses or administering CsA a longer duration. MATERIALS AND METHODS Male Sprague-Dawley rats (6 per group) were anesthetized with halothane and the left renal pedicle clamped for 30 minutes through a flank incision. Control animals were sham operated. The animals were injected daily intraperitoneally either with CsA (15 mg/kg) or drug vehicle for 30 days. Kidney sections were stained with Masson’s trichrome for fibrosis scoring (0 to 3), hematoxylin and eosin for histologic scoring (tubular dilatation and inflammatory infiltrate), and PAS for arteriolar hyalinosis. For immunohistochemical analysis, sections were dewaxed and hydrated in graded ethanol, treated with 3% H2O2 (10 minutes) to quench endogenous peroxidase activity, 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(02)03869-1 174
treated with 0.1% trypsin at 37°C (10 minutes), and incubated (30 minutes) in diluted blocking serum to suppress nonspecific binding of IgG. Sections were incubated at 4°C overnight with primary antibodies to collagen III 1:10, collagen IV 1:35, ␣-SMA 1:100, EDI (macrophage marker) 1:50, and TGF1 1:50. Sections were then incubated (30 minutes) with a biotin-conjugated secondary antibody followed by an avidin-biotin reagent (30 minutes), developed using a commercial AEC kit and counterstained with hematoxylin.Staining was quantified by point counting. The results, expressed as mean ⫾ SEM, were compared using Mann-Whitney test (SPSS). Differences were considered significant when P ⬍ .05.
RESULTS
RWI alone produced no significant effect on the development of renal fibrosis or injury. However, when RWI was induced in CsA-treated rats, there was a significant increase in the fibrosis score (1.96 ⫾ 0.49) compared to either renal warm ischaemia alone (0.31 ⫾ 0.14, P ⬍ .05) or CsA alone (0.31 ⫾ 0.07, P ⬍ .05). Both the tubular dilatation score and the inflammatory infiltrate score were significantly higher among CsA-treated ischemic kidneys (1.09 ⫾ 0.33 and 2.2 ⫾ 0.43, respectively) compared to either CsA alone (0.27 ⫾ 0.09, P ⬍ .05 and 0.4 ⫾ 0.15, P ⬍ .05 respectively) or ischemia alone (0, P ⬍ .01 and 0.5 ⫾ 0.14, P ⬍ .05, respectively). All CsA-treated animals showed a significant increase in arteriolar hyalinosis (47 ⫾ 5%) compared to 30 minutes of RWI alone (0%, P ⬍ .01) or vehicle-treated, sham-operated animals (0%, P ⬍ .01) in which cases it remained undetectable. There was a significant increase in ␣-SMA immunostaining when ischemia was induced in CsA-treated kidneys (21.0 ⫾ 6.4) compared to ischemia alone (2.3 ⫾ 0.5, P ⬍ .01) or CsA alone (6.0 ⫾ 3.2, P ⬍ .05). Both TGF-1 immunostaining and the number ED1 positive cells per field were significantly higher in CsA-treated ischemic kidneys (39.5 ⫾ 7.3% and 64.9 ⫾ 15.4 per field, respectively) compared to either 30 minutes of RWI alone (5.9 ⫾ 0.8%, P ⬍ .01 and 4.9 ⫾ 2.6, P ⬍ .01 per field, respectively) or CsA alone (10.5 ⫾ 3.7%, P ⬍ .05 and 1.8 ⫾ 0.6, P ⬍ .01 per field, From the Sheffield Kidney Institute, Northern General Hospital, Sheffield, UK. Supported by the Sheffield Kidney Patients Association. Address reprint requests to Mr A.M.K. Ahmed, Sheffield Kidney Institute, Northern General Hospital, Sheffield, S5 7AU, UK. © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 174 –175 (2003)
CHRONIC CSA NEPHROTOXICITY
respectively). Collagen type III and type IV immunostaining were significantly increased in CsA-treated kidneys (37.9 ⫾ 12.1 and 58.1 ⫾ 3.1, respectively) compared to either ischemia alone (8.46 ⫾ 3, P ⬍ .05 and 39 ⫾ 4.9, P ⬍ .05, respectively) or CsA alone (10.0 ⫾ 2.5, P ⬍ .05 and 36.3 ⫾ 4.0, P ⬍ .05, respectively). There was no difference in CsA levels between the groups treated with this drug. DISCUSSION
This study demonstrates a potentiation effect of a short period of renal warm ischemia on cyclosporine nephrotoxicity, expressed as a significant increase in the fibrosis markers (fibrosis score, collagen III and collagen IV), cellular infiltration and expression of the fibrogenic cytokine TGF-1. Our findings of significant macrophage infiltration in CsA-induced nephrotoxicity correlate with Vieira et al.7 who also demonstrated that the interstitial macrophage infiltration was progressive with CsA administration and ultimately correlated with the fibrotic areas. Employing higher doses of CsA, Young et al.5 also described similar findings concerning an early role of macrophages. Macrophages probably are involved in cytokine production and certainly in the interstitial fibrogenic process. These cells may be attracted to renal tissue by many mediators, such as osteopontin and angiotensin II.8 Angiotensin II may be the link between CsA and macrophage infiltration, because blockade of the renin-angiotensin system reduces CsAinduced interstitial fibrosis.9 We suggest that TGF-1 might be secreted by infiltrating inflammatory cells. Indeed, in vitro studies show CsA to directly stimulate TGF-1 transcription in T cells in vitro10 and TGF-1 synthesis in tubular cells and fibroblasts,11 whereas in vivo the expression of TGF-1 is increased in human mononuclear cells.12 TGF-1 has been reported to be a mediator of the fibrogenic effects of local angiotensin II action.13 It has been also demonstrated that angiotensin
175
II antagonists prevent TGF- expression and fibrosis development in CsA-treated rats.12 CsA-treated rats also show a progressive increase in mRNA expression of TGF-1 and matrix proteins. It has been proposed that the fibrosis during chronic CsA nephropathy involves a dual action of TGF1 on matrix deposition and degradation.14 In conclusion, by the use of a short period of renal warm ischemia, we have developed a model of CsA-induced nephrotoxicity that mimics the clinical situation without the use of higher doses or a longer period of drug administration. This model offers the opportunity to test the therapeutic interventions that might block or ameliorate the nephrotoxic effect of this drug.
REFERENCES 1. Sund S, Forre O, Berg KJ, et al: Clin Nephrol 41:33, 1994 2. Goldstein DJ, Zuech N, Sehgal V, et al: Transplantation 15:664, 1997 3. Bennett EM: Int J Clin Pharmacol Ther 34:515, 1996 4. Bertani T, Ferrazzi P, Schieppati A, et al: Kidney Int 40:243, 1991 5. Young BA, Burdmann EA, Johnson RJ, et al: Kidney Int 48:439, 1995 6. Elzinga LW, Rosen S, Bennett WM, et al: J Am Soc Nephrol 4:214, 1993 7. Vieira JM, Noronha IL, Malheiros DM, et al: Transplantation 68:1746, 1999 8. Pichler RH, Franceschini N, Young BA: J Am Soc Nephrol 6:1186, 1995 9. Kon Y, Endoch D, Murakami K, et al: Ant Rec 241:451, 1995 10. Prashar Y, Khanna A, Sehajpal,, et al: FEBS Lett 23:109, 1995 11. Wolf G, Ziyadeh FN, Helmchen U, et al: Am J Physiol 246:F940, 1995 12. Shin GT, Khanna A, Ding R, et al: Transplantation 15:313, 1998 13. Shihab FS, Bennett WM, Tanner AM, et al: Kidney Int 25:660, 1997 14. Shihab FS, Andoh TF, Tanner AM, et al: Kidney Int 49:1141, 1996
S
INCE ITS introduction in 1976, cyclosporine (CsA) has markedly improved the survival of solid organ transplants and has also been beneficial for the treatment of autoimmune diseases. However, the benefits of CsA therapy have been potentially offset by the occurrence of nephrotoxicity. CsA nephrotoxicity which has been reported since the early 1980s, occurs even when low doses of the drug are used in the treatment of autoimmune disease.1 In the setting of cardiac transplantation, CsA nephrotoxicity has been related to the development of end-stage renal failure in about 10% of patients.2 The contribution of CsA nephrotoxicity to chronic allograft dysfunction is hard to determine due to many confounding etiologic factors, including ischemia/reperfusion injury, chronic rejection, and de novo nephropathies.3 Chronic renal injury induced by CsA is characterized by irreversible striped interstitial fibrosis and afferent arteriolar hyalinosis.3 The mechanisms of CsA-induced renal fibrosis are not well understood; they have been attributed to sustained afferent arteriolar vasoconstriction and arteriopathy, leading to preglomerular ischemia.4 The lesion of chronic CsA nephropathy has been difficult to reproduce in animals. Thus, the pathogenesis of this type of CsA-induced renal injury has been difficult to study.5 An animal model of chronic CsA nephrotoxicity has been described using salt depletion as stimulus to accelerate the long-term effects of CsA treatment.6 In the present study, the long-term effects of CsA treatment were potentiated by a stimulus of 30 minutes of warm ischemia (RWI). The aim of this comparative histologic and morphologic study was to introduce a model of CsA nephrotoxicity without using either higher doses or administering CsA a longer duration. MATERIALS AND METHODS Male Sprague-Dawley rats (6 per group) were anesthetized with halothane and the left renal pedicle clamped for 30 minutes through a flank incision. Control animals were sham operated. The animals were injected daily intraperitoneally either with CsA (15 mg/kg) or drug vehicle for 30 days. Kidney sections were stained with Masson’s trichrome for fibrosis scoring (0 to 3), hematoxylin and eosin for histologic scoring (tubular dilatation and inflammatory infiltrate), and PAS for arteriolar hyalinosis. For immunohistochemical analysis, sections were dewaxed and hydrated in graded ethanol, treated with 3% H2O2 (10 minutes) to quench endogenous peroxidase activity, 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(02)03869-1 174
treated with 0.1% trypsin at 37°C (10 minutes), and incubated (30 minutes) in diluted blocking serum to suppress nonspecific binding of IgG. Sections were incubated at 4°C overnight with primary antibodies to collagen III 1:10, collagen IV 1:35, ␣-SMA 1:100, EDI (macrophage marker) 1:50, and TGF1 1:50. Sections were then incubated (30 minutes) with a biotin-conjugated secondary antibody followed by an avidin-biotin reagent (30 minutes), developed using a commercial AEC kit and counterstained with hematoxylin.Staining was quantified by point counting. The results, expressed as mean ⫾ SEM, were compared using Mann-Whitney test (SPSS). Differences were considered significant when P ⬍ .05.
RESULTS
RWI alone produced no significant effect on the development of renal fibrosis or injury. However, when RWI was induced in CsA-treated rats, there was a significant increase in the fibrosis score (1.96 ⫾ 0.49) compared to either renal warm ischaemia alone (0.31 ⫾ 0.14, P ⬍ .05) or CsA alone (0.31 ⫾ 0.07, P ⬍ .05). Both the tubular dilatation score and the inflammatory infiltrate score were significantly higher among CsA-treated ischemic kidneys (1.09 ⫾ 0.33 and 2.2 ⫾ 0.43, respectively) compared to either CsA alone (0.27 ⫾ 0.09, P ⬍ .05 and 0.4 ⫾ 0.15, P ⬍ .05 respectively) or ischemia alone (0, P ⬍ .01 and 0.5 ⫾ 0.14, P ⬍ .05, respectively). All CsA-treated animals showed a significant increase in arteriolar hyalinosis (47 ⫾ 5%) compared to 30 minutes of RWI alone (0%, P ⬍ .01) or vehicle-treated, sham-operated animals (0%, P ⬍ .01) in which cases it remained undetectable. There was a significant increase in ␣-SMA immunostaining when ischemia was induced in CsA-treated kidneys (21.0 ⫾ 6.4) compared to ischemia alone (2.3 ⫾ 0.5, P ⬍ .01) or CsA alone (6.0 ⫾ 3.2, P ⬍ .05). Both TGF-1 immunostaining and the number ED1 positive cells per field were significantly higher in CsA-treated ischemic kidneys (39.5 ⫾ 7.3% and 64.9 ⫾ 15.4 per field, respectively) compared to either 30 minutes of RWI alone (5.9 ⫾ 0.8%, P ⬍ .01 and 4.9 ⫾ 2.6, P ⬍ .01 per field, respectively) or CsA alone (10.5 ⫾ 3.7%, P ⬍ .05 and 1.8 ⫾ 0.6, P ⬍ .01 per field, From the Sheffield Kidney Institute, Northern General Hospital, Sheffield, UK. Supported by the Sheffield Kidney Patients Association. Address reprint requests to Mr A.M.K. Ahmed, Sheffield Kidney Institute, Northern General Hospital, Sheffield, S5 7AU, UK. © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 174 –175 (2003)
CHRONIC CSA NEPHROTOXICITY
respectively). Collagen type III and type IV immunostaining were significantly increased in CsA-treated kidneys (37.9 ⫾ 12.1 and 58.1 ⫾ 3.1, respectively) compared to either ischemia alone (8.46 ⫾ 3, P ⬍ .05 and 39 ⫾ 4.9, P ⬍ .05, respectively) or CsA alone (10.0 ⫾ 2.5, P ⬍ .05 and 36.3 ⫾ 4.0, P ⬍ .05, respectively). There was no difference in CsA levels between the groups treated with this drug. DISCUSSION
This study demonstrates a potentiation effect of a short period of renal warm ischemia on cyclosporine nephrotoxicity, expressed as a significant increase in the fibrosis markers (fibrosis score, collagen III and collagen IV), cellular infiltration and expression of the fibrogenic cytokine TGF-1. Our findings of significant macrophage infiltration in CsA-induced nephrotoxicity correlate with Vieira et al.7 who also demonstrated that the interstitial macrophage infiltration was progressive with CsA administration and ultimately correlated with the fibrotic areas. Employing higher doses of CsA, Young et al.5 also described similar findings concerning an early role of macrophages. Macrophages probably are involved in cytokine production and certainly in the interstitial fibrogenic process. These cells may be attracted to renal tissue by many mediators, such as osteopontin and angiotensin II.8 Angiotensin II may be the link between CsA and macrophage infiltration, because blockade of the renin-angiotensin system reduces CsAinduced interstitial fibrosis.9 We suggest that TGF-1 might be secreted by infiltrating inflammatory cells. Indeed, in vitro studies show CsA to directly stimulate TGF-1 transcription in T cells in vitro10 and TGF-1 synthesis in tubular cells and fibroblasts,11 whereas in vivo the expression of TGF-1 is increased in human mononuclear cells.12 TGF-1 has been reported to be a mediator of the fibrogenic effects of local angiotensin II action.13 It has been also demonstrated that angiotensin
175
II antagonists prevent TGF- expression and fibrosis development in CsA-treated rats.12 CsA-treated rats also show a progressive increase in mRNA expression of TGF-1 and matrix proteins. It has been proposed that the fibrosis during chronic CsA nephropathy involves a dual action of TGF1 on matrix deposition and degradation.14 In conclusion, by the use of a short period of renal warm ischemia, we have developed a model of CsA-induced nephrotoxicity that mimics the clinical situation without the use of higher doses or a longer period of drug administration. This model offers the opportunity to test the therapeutic interventions that might block or ameliorate the nephrotoxic effect of this drug.
REFERENCES 1. Sund S, Forre O, Berg KJ, et al: Clin Nephrol 41:33, 1994 2. Goldstein DJ, Zuech N, Sehgal V, et al: Transplantation 15:664, 1997 3. Bennett EM: Int J Clin Pharmacol Ther 34:515, 1996 4. Bertani T, Ferrazzi P, Schieppati A, et al: Kidney Int 40:243, 1991 5. Young BA, Burdmann EA, Johnson RJ, et al: Kidney Int 48:439, 1995 6. Elzinga LW, Rosen S, Bennett WM, et al: J Am Soc Nephrol 4:214, 1993 7. Vieira JM, Noronha IL, Malheiros DM, et al: Transplantation 68:1746, 1999 8. Pichler RH, Franceschini N, Young BA: J Am Soc Nephrol 6:1186, 1995 9. Kon Y, Endoch D, Murakami K, et al: Ant Rec 241:451, 1995 10. Prashar Y, Khanna A, Sehajpal,, et al: FEBS Lett 23:109, 1995 11. Wolf G, Ziyadeh FN, Helmchen U, et al: Am J Physiol 246:F940, 1995 12. Shin GT, Khanna A, Ding R, et al: Transplantation 15:313, 1998 13. Shihab FS, Bennett WM, Tanner AM, et al: Kidney Int 25:660, 1997 14. Shihab FS, Andoh TF, Tanner AM, et al: Kidney Int 49:1141, 1996