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Vascular endothelin, TGF–beta, and PDGF expression in FK506 nephrotoxicity of rats
Vascular Endothelin, TGF–Beta, and PDGF Expression in FK506 Nephrotoxicity of Rats H.J. Jeong, Y.S. Kim, and I.C. Hong
F
K506 IS A macrolide immunosuppressant, exerting an immunosuppressive action by inhibiting the activation process of T cells, the way cyclosporine (CyA) does. Complications associated with FK506 are similar to those seen in CyA, including nephrotoxicity, both functional or morphological. Morphological changes include hyperplasia of the juxtaglomerular apparatus (JGA), vacuolization, and microcalcification of renal tubules, interstitial fibrosis, and microvascular changes.1,2 Vasoconstriction by FK506 treatment may be attributed either to direct toxicity to endothelium or to secretion of mediators related to vascular constriction. Such mediators are endothelin, renin, prostanoids, and nitric oxide. Endothelin is known as the most potent vasoconstrictor. Its secretion is increased not only in cultured endothelial cells, but also in mesangial cells and in tubular cells with FK506 treatment.3–5 Activation of renin angiotensin system is another candidate, which is supported by hyperplasia of JGA with increased renin immunoreactivity.1 If vasoconstriction is an early event leading to reduction of glomerular filtration rate, thickening and narrowing of microvasculature is a late sequelae, for which growth factors are partly responsible. Shehata et al6 –7 demonstrated PDGF and TGF– beta immunoreactivity in afferent arterioles of rats with CyA treatment and suggested a role for PDGF and TGF– beta in the pathogenesis of CyA toxicity. Although FK506 shares much similarity with CyA, the incidence of hypertension and hypercholesterolemia is less in cases of patients treated with FK506. Moreover, therapeutic doses of CyA, unlike FK506, induces the synthesis of endothelin in human endothelial cells,3 which may affect different expression of growth factors and magnitude of vascular involvement. However, histopathological studies comparing FK506 with CyA did not reveal any significant difference in the incidence or in the pattern of vascular injury, which raises a suspicion that it may be related to other factors such as renal ischemia. The present study was, thus, conducted to investigate the vascular expressions of endothelin, PDGF, and TGF– beta in rats suffering FK506 nephrotoxicity and also to evaluate the effect of ischemia in this model. MATERIALS AND METHODS Male Sprague Dawley rats weighing 200 to 250 gm were used. The rats in the experimental group received FK506 (gift from Fujisawa
Pharmaceutical Co Ltd, Osaka, Japan), and the control rats received the same volume of physiological saline. FK506 powder was dissolved in saline and was given daily at a dosage of 2mg/kg by intramuscular route for 4 weeks at maximum. Five rats were sacrificed 3 days, 1 week, 2 weeks, and 4 weeks after the initiation of the study, respectively. Renal ischemia was induced by occluding the left renal artery for 45 minutes, and rats were sacrificed 3 days, 1 week, and 2 weeks after reperfusion. Kidneys were processed for light microscopy and stained with hematoxylin eosin and periodic acid–Schiff (PAS) methods. For immunohistochemistry, formalin fixed and paraffin embedded renal tissue was stained with antibodies against endothelin (Affinity Bioreagents, Co, USA), TGF-betal (Santa Cruz, Calif, USA), and PDGF (Oncogene Science, NY, USA) using the ABC method. The number of juxtaglomerular apparatuses and arterioles positive for each antibody was counted in at least 20 fields under x200 magnification and was expressed as mean 6 S.D. per mm2 cortex. Statistical analysis among groups was done using two–way ANOVA. A P value less than 0.05 was considered significant.
RESULTS
The pathological findings of FK506 toxicity could be seen in tubules and microvasculature, without accompanying interstitial fibrosis. Proximal tubules showed coarse vacuolar change and many lysosomes in the cytoplasm. JGA and afferent arterioles were prominent with PAS positive granules, which was extended proximally to interlobular arteries with increased duration of FK506 treatment. With increasing duration, TGF– beta reactivity was increased in afferent arterioles, and the increase in reactivity was statistically significant, which roughly paralleled PAS positivity. However, no such results were shown in cases of PDGF and endothelin. Renal ischemia induced acute tubular necrosis in 3 days, and the recovery was delayed as long as 2 weeks with FK506 treatment. Ischemia itself increased vascular TGF– beta as well as endothelin and PDGF reactivities, which was statisFrom the Department of Pathology and Surgery, College of Medicine, Institute of Renal Disease, Yonsei University, Seoul, Korea. This work was supported by a Yonsei University Faculty Research Fund, 1995. Address reprint requests to Dr Hyeon Joo Jeong, College of Medicine, Institute of Renal Diseases, Yonsei University, Seoul, Korea.
0041-1345/98/$19.00 PII S0041-1345(98)01152-X
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
3596
Transplantation Proceedings, 30, 3596–3597 (1998)
FK506 NEPHROTOXICITY OF RATS
3597
Table 1. Immunoreactivity of Juxtaglomerular Apparatus and Afferent Arterioles in FK506 Toxicity of Rats Duration Stain/antisera
PAS
TGF beta
PDGF
endothelin
Group
Control FK506*† FK506 1 Control FK506*† FK506 1 Control FK506† FK506 1 Control FK506† FK506 1
ischemia
ischemia
ischemia
ischemia
3d
1 wk
2 wk
0.5 6 0.1 0.6 6 0.2 0.2 6 0.2 0.8 6 0.9 0.2 6 0.4 1.7 6 0.6 0.0 6 0.0 0.0 6 0.0 1.6 6 1.3 0.0 6 0.0 0.0 6 0.0 0.1 6 0.1
0.7 6 0.4 1.9 6 0.8 0.6 6 0.4 1.2 6 0.7 1.1 6 0.2 2.3 6 0.7 0.0 6 0.0 0.0 6 0.0 0.8 6 0.8 0.0 6 0.0 0.3 6 0.5 0.5 6 0.3
0.9 6 0.5 1.9 6 0.7 0.6 6 0.4 0.9 6 0.1 2.0 6 1.2 2.1 6 0.8 0.0 6 0.0 0.0 6 0.0 0.6 6 0.6 0.0 6 0.0 0.0 6 0.0 0.4 6 0.1
*P , .05 as compared with control. † P , .05 as compared with FK506 1 ischemia.
tically significant. Renal ischemia in FK506 treated rats further upregulated the expression of these markers in a similar distribution. However, the expression of endothelin was rare and weak in arterioles, and mostly found in endothelial cells of peritubular and glomerular capillaries in rats sacrificed 1 and 2 weeks after reperfusion. PAS staining was decreased in ischemic kidneys regardless of FK506 treatment (Table 1). DISCUSSION
Assuming that similar factors were involved in the microvascular injury of FK506 as in CyA, it was not surprising to observe vascular expression of endothelin, TGF– beta, and PDGF in FK506 nephrotoxicity. TGF– beta expression was increased in JGA and in afferent arterioles with increased duration of FK506 treatment and with ischemia, which was statistically significant. Increased PDGF expression superimposed on ischemia differed from that of CyA,6 for which PDGF was expressed even without ischemia. It might be attributed to the difference of commercial antibodies used; however, PDGF might play a minor role in the pathogenesis of FK506 toxicity unless accompanied by ischemia. Both growth factors might play a role in vascular wall narrowing and thickening, especially upon ischemia, independently or synergistically, either by stimulating renin secretion or by smooth muscle proliferation. Endothelin also was expressed upon ischemia, but mostly in the peritubular and glomerular capillaries unlike TGF– beta or PDGF. Nevertheless, endothelin certainly had an additive effect on microvascular injury. FK506 treatment increased granularity of JGA and afferent arterioles by PAS stain, but decreased granularity with ischemia. PAS positive granules had the same location as renin, which was presumed to be
stored in an inactive state, and to be secreted as both prorenin and active renin.8 Decreased PAS granularity, thus might indicate either increased secretion and/or decreased synthesis of renin with ischemia. A complex interrelationship exists between renin, TGF– beta, and PDGF, all of which are confined within the juxtaglomerular cells of kidneys6 –7 and all may contribute to vascular pathology. In conclusion, FK506 toxicity was comparable to CyA toxicity. It mostly affected JGA and afferent arterioles, and it showed increased PAS and TGF– beta reactivity. However, decreased expression of PDGF might be an explanation for less severe vascular damage in FK506 nephrotoxicity. Since expression levels of endothelin, TGF– beta, and PDGF were increased in ischemic kidneys, it might be helpful to prevent ischemic damage as well as to hinder secretion of these factors in order to reduce FK506 nephrotoxicity. REFERENCES 1. Yamada K, Sugisaki Y, Akimoto M, et al: Transplant Proc 23:3130, 1991 2. Stillman IE, Andoh TF, Burdmann EA, et al: Lab Invest 73:794, 1995 3. Takeda Y, Yoneda T, Ito Y, et al: J Cardiovasc Pharmacol 22(suppl 8):S310, 1993 4. Goodall T, Kind CN, Hammond TG: J Cardiovasc Pharmacol 26(suppl 3):S482, 1995 5. Moutabarrik A, Ishibashi M, Fukunaga M, et aI: Transplant Proc 23:3133, 1991 6. Shehata M, el Nahas AM, Barkworth E, et al: Kidney Int 46:726, 1994 7. Shehata M, Cope GH, Johnson TS, et al: Kidney Int 48:1487, 1995 8. Lee BN: Kidney Int 52:248, 1997
F
K506 IS A macrolide immunosuppressant, exerting an immunosuppressive action by inhibiting the activation process of T cells, the way cyclosporine (CyA) does. Complications associated with FK506 are similar to those seen in CyA, including nephrotoxicity, both functional or morphological. Morphological changes include hyperplasia of the juxtaglomerular apparatus (JGA), vacuolization, and microcalcification of renal tubules, interstitial fibrosis, and microvascular changes.1,2 Vasoconstriction by FK506 treatment may be attributed either to direct toxicity to endothelium or to secretion of mediators related to vascular constriction. Such mediators are endothelin, renin, prostanoids, and nitric oxide. Endothelin is known as the most potent vasoconstrictor. Its secretion is increased not only in cultured endothelial cells, but also in mesangial cells and in tubular cells with FK506 treatment.3–5 Activation of renin angiotensin system is another candidate, which is supported by hyperplasia of JGA with increased renin immunoreactivity.1 If vasoconstriction is an early event leading to reduction of glomerular filtration rate, thickening and narrowing of microvasculature is a late sequelae, for which growth factors are partly responsible. Shehata et al6 –7 demonstrated PDGF and TGF– beta immunoreactivity in afferent arterioles of rats with CyA treatment and suggested a role for PDGF and TGF– beta in the pathogenesis of CyA toxicity. Although FK506 shares much similarity with CyA, the incidence of hypertension and hypercholesterolemia is less in cases of patients treated with FK506. Moreover, therapeutic doses of CyA, unlike FK506, induces the synthesis of endothelin in human endothelial cells,3 which may affect different expression of growth factors and magnitude of vascular involvement. However, histopathological studies comparing FK506 with CyA did not reveal any significant difference in the incidence or in the pattern of vascular injury, which raises a suspicion that it may be related to other factors such as renal ischemia. The present study was, thus, conducted to investigate the vascular expressions of endothelin, PDGF, and TGF– beta in rats suffering FK506 nephrotoxicity and also to evaluate the effect of ischemia in this model. MATERIALS AND METHODS Male Sprague Dawley rats weighing 200 to 250 gm were used. The rats in the experimental group received FK506 (gift from Fujisawa
Pharmaceutical Co Ltd, Osaka, Japan), and the control rats received the same volume of physiological saline. FK506 powder was dissolved in saline and was given daily at a dosage of 2mg/kg by intramuscular route for 4 weeks at maximum. Five rats were sacrificed 3 days, 1 week, 2 weeks, and 4 weeks after the initiation of the study, respectively. Renal ischemia was induced by occluding the left renal artery for 45 minutes, and rats were sacrificed 3 days, 1 week, and 2 weeks after reperfusion. Kidneys were processed for light microscopy and stained with hematoxylin eosin and periodic acid–Schiff (PAS) methods. For immunohistochemistry, formalin fixed and paraffin embedded renal tissue was stained with antibodies against endothelin (Affinity Bioreagents, Co, USA), TGF-betal (Santa Cruz, Calif, USA), and PDGF (Oncogene Science, NY, USA) using the ABC method. The number of juxtaglomerular apparatuses and arterioles positive for each antibody was counted in at least 20 fields under x200 magnification and was expressed as mean 6 S.D. per mm2 cortex. Statistical analysis among groups was done using two–way ANOVA. A P value less than 0.05 was considered significant.
RESULTS
The pathological findings of FK506 toxicity could be seen in tubules and microvasculature, without accompanying interstitial fibrosis. Proximal tubules showed coarse vacuolar change and many lysosomes in the cytoplasm. JGA and afferent arterioles were prominent with PAS positive granules, which was extended proximally to interlobular arteries with increased duration of FK506 treatment. With increasing duration, TGF– beta reactivity was increased in afferent arterioles, and the increase in reactivity was statistically significant, which roughly paralleled PAS positivity. However, no such results were shown in cases of PDGF and endothelin. Renal ischemia induced acute tubular necrosis in 3 days, and the recovery was delayed as long as 2 weeks with FK506 treatment. Ischemia itself increased vascular TGF– beta as well as endothelin and PDGF reactivities, which was statisFrom the Department of Pathology and Surgery, College of Medicine, Institute of Renal Disease, Yonsei University, Seoul, Korea. This work was supported by a Yonsei University Faculty Research Fund, 1995. Address reprint requests to Dr Hyeon Joo Jeong, College of Medicine, Institute of Renal Diseases, Yonsei University, Seoul, Korea.
0041-1345/98/$19.00 PII S0041-1345(98)01152-X
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
3596
Transplantation Proceedings, 30, 3596–3597 (1998)
FK506 NEPHROTOXICITY OF RATS
3597
Table 1. Immunoreactivity of Juxtaglomerular Apparatus and Afferent Arterioles in FK506 Toxicity of Rats Duration Stain/antisera
PAS
TGF beta
PDGF
endothelin
Group
Control FK506*† FK506 1 Control FK506*† FK506 1 Control FK506† FK506 1 Control FK506† FK506 1
ischemia
ischemia
ischemia
ischemia
3d
1 wk
2 wk
0.5 6 0.1 0.6 6 0.2 0.2 6 0.2 0.8 6 0.9 0.2 6 0.4 1.7 6 0.6 0.0 6 0.0 0.0 6 0.0 1.6 6 1.3 0.0 6 0.0 0.0 6 0.0 0.1 6 0.1
0.7 6 0.4 1.9 6 0.8 0.6 6 0.4 1.2 6 0.7 1.1 6 0.2 2.3 6 0.7 0.0 6 0.0 0.0 6 0.0 0.8 6 0.8 0.0 6 0.0 0.3 6 0.5 0.5 6 0.3
0.9 6 0.5 1.9 6 0.7 0.6 6 0.4 0.9 6 0.1 2.0 6 1.2 2.1 6 0.8 0.0 6 0.0 0.0 6 0.0 0.6 6 0.6 0.0 6 0.0 0.0 6 0.0 0.4 6 0.1
*P , .05 as compared with control. † P , .05 as compared with FK506 1 ischemia.
tically significant. Renal ischemia in FK506 treated rats further upregulated the expression of these markers in a similar distribution. However, the expression of endothelin was rare and weak in arterioles, and mostly found in endothelial cells of peritubular and glomerular capillaries in rats sacrificed 1 and 2 weeks after reperfusion. PAS staining was decreased in ischemic kidneys regardless of FK506 treatment (Table 1). DISCUSSION
Assuming that similar factors were involved in the microvascular injury of FK506 as in CyA, it was not surprising to observe vascular expression of endothelin, TGF– beta, and PDGF in FK506 nephrotoxicity. TGF– beta expression was increased in JGA and in afferent arterioles with increased duration of FK506 treatment and with ischemia, which was statistically significant. Increased PDGF expression superimposed on ischemia differed from that of CyA,6 for which PDGF was expressed even without ischemia. It might be attributed to the difference of commercial antibodies used; however, PDGF might play a minor role in the pathogenesis of FK506 toxicity unless accompanied by ischemia. Both growth factors might play a role in vascular wall narrowing and thickening, especially upon ischemia, independently or synergistically, either by stimulating renin secretion or by smooth muscle proliferation. Endothelin also was expressed upon ischemia, but mostly in the peritubular and glomerular capillaries unlike TGF– beta or PDGF. Nevertheless, endothelin certainly had an additive effect on microvascular injury. FK506 treatment increased granularity of JGA and afferent arterioles by PAS stain, but decreased granularity with ischemia. PAS positive granules had the same location as renin, which was presumed to be
stored in an inactive state, and to be secreted as both prorenin and active renin.8 Decreased PAS granularity, thus might indicate either increased secretion and/or decreased synthesis of renin with ischemia. A complex interrelationship exists between renin, TGF– beta, and PDGF, all of which are confined within the juxtaglomerular cells of kidneys6 –7 and all may contribute to vascular pathology. In conclusion, FK506 toxicity was comparable to CyA toxicity. It mostly affected JGA and afferent arterioles, and it showed increased PAS and TGF– beta reactivity. However, decreased expression of PDGF might be an explanation for less severe vascular damage in FK506 nephrotoxicity. Since expression levels of endothelin, TGF– beta, and PDGF were increased in ischemic kidneys, it might be helpful to prevent ischemic damage as well as to hinder secretion of these factors in order to reduce FK506 nephrotoxicity. REFERENCES 1. Yamada K, Sugisaki Y, Akimoto M, et al: Transplant Proc 23:3130, 1991 2. Stillman IE, Andoh TF, Burdmann EA, et al: Lab Invest 73:794, 1995 3. Takeda Y, Yoneda T, Ito Y, et al: J Cardiovasc Pharmacol 22(suppl 8):S310, 1993 4. Goodall T, Kind CN, Hammond TG: J Cardiovasc Pharmacol 26(suppl 3):S482, 1995 5. Moutabarrik A, Ishibashi M, Fukunaga M, et aI: Transplant Proc 23:3133, 1991 6. Shehata M, el Nahas AM, Barkworth E, et al: Kidney Int 46:726, 1994 7. Shehata M, Cope GH, Johnson TS, et al: Kidney Int 48:1487, 1995 8. Lee BN: Kidney Int 52:248, 1997