Inhibition of the Renin Angiotensin System Decreases Fibrogenic Cytokine Expression in Tacrolimus Nephrotoxicity in Rats

Inhibition of the Renin Angiotensin System Decreases Fibrogenic Cytokine Expression in Tacrolimus Nephrotoxicity in Rats H. Deniz, B. Ög˘ütmen, F. Çakalag˘aog˘lu, S. Tug˘lular, Ç. Özener, and E. Akog˘lu ABSTRACT The aim of our study was to investigate the influence of angiotensin-converting enzyme (ACE) inhibition and angiotensin II receptor blockage on the renal function by light microscopic and immunohistochemical findings in a rat model of tacrolimus nephrotoxicity. Thirty-two male Wistar rats were divided into four groups of eight: G1 ⫽ control group; G2-G3, G4 ⫽ Tacrolimus (Tac) 1 mg/kg/d intraperitoneally (ip); G3 (Tac ⫹ Q) ⫽ ip Tac and peroral quinapril 10 mg/kg; and G4 (Tac ⫹ V) ⫽ Tac and valsartan 40 mg/d. Serum blood urea nitrogen (BUN), creatinine, and creatinine clearance were measured before and at the end of the study period. Renal tissues were assessed for light microscopic findings of tacrolimus toxicity. Transforming growth factor-␤, VEGF, PDGF, BMP-7, and interleukin-6 (IL-6) expression were semiquantitatively scored after immunohistochemical staining. At the end of the study period serum BUN and creatinine levels were increased in all groups, but creatinine clearance was not significantly changed between the groups. Afferent arteriolopathy was significantly less pronounced in G3 versus G2 and G4. Interstial fibrosis was significantly less pronounced in G3 and G4 versus G2. TGF-␤, PDGF, and IL-6 expression were significantly increased in G2, G3, and G4 compared to G1, and in G2 compared to G3 and G4. BMP-7 expression was significantly decreased in G2, G3, and G4 compared to G1, whereas the differences between G2, G3, and G4 failed to reach statistical significance. In conclusion, the results of our study suggested that renin angiotensin inhibition down-regulates fibrogenic cytokine expression in rats displaying tacrolimus nephrotoxicity.

T

ACROLIMUS (Tac) is a calcineurin inhibitor used as an immunosuppresive agent following transplantation.1 Substantial evidence suggests that it reduces the risk of acute rejection episodes2,3 but chronic nephrotoxicity associated with long-term Tac uses limits its use in both renal and nonrenal allograft recipients as well as in patients with autoimmune diseases.4 Chronic Tac toxicity results in structural and functional impairment in the kidneys.5 The pathogenesis of chronic Tac nephrotoxicity has not been completely elucidated but seems to be associated with up-regulated transforming growth factor-␤ (TGF-␤) and platelet derived growth factor (PDGF) production.6,7 Angiotensin II, a renal growth factor, activates mesangial and tubular cells as well as interstitial fibroblasts.8 –10 Some of these effects seem to be mediated by the release of other growth factors and cytokines, such as TGF-␤,11–13 VEGF,14 –16 PDGF,17 and interleukin-6 (IL-6).18,19 Bone morphogenic protein-7 (BMP-7) is a member of TGF family, which © 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 38, 483– 486 (2006)

counteracts some of the profibrogenic actions of TGF-␤.20 The use of ACE inhibitors (ACEI) and angiotensin II receptor blockage (AIIRB) has been previously shown to ameliorate proteinuria, inflammatory cell infiltration, and fibrosis in several models of kidney damage.21 The aim of our study was to evaluate the roles of TGF-␤, PDGF, VEGF, IL-6, and BMP-7 expression in the patho-

From the Department of Internal Medicine (H.D.), Division of Nephrology (B.Ö., S.T., Ç.Ö., E.A.), and Department of Pathology (F.Ç.), Marmara University Medical School, Istanbul, Turkey. This study was supported by a limited educational grant provided by Marmara University Investigation Fund and study medications were provided by Pfizer (quinapril tb), Novartis (valsartan tb), and tacrolimus amp was provided by Fujisawa, Japan. Address reprint requests to Dr Serhan Tug˘lular, Hemodialysis Unit, Marmara University Hospital, Altunizade, Istanbul, Turkey. E-mail: [email protected] 0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2005.12.056 483

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Table 1. Serum Creatinine and BUN Levels and Creatinine Clearances Groups

Cr-B

Cr-A

BUN-B

BUN-A

Cr Cl-B

Cr Cl-A

G1 G2 G3 G4

0.24 ⫾ 0.2 0.65 ⫾ 0.2 0.30 ⫾ 0.17 0.22 ⫾ 0.2

0.35 ⫾ 0.2 0.57 ⫾ 0.14 0.38 ⫾ 0.2 0.44 ⫾ 0.11

15.14 ⫾ 2.19 39.48 ⫾ 5.63 15.25 ⫾ 2.71 16.71 ⫾ 1.60

20.71 ⫾ 2.69 55.6 ⫾ 9.13 40.00 ⫾ 28.95 50.71 ⫾ 33.79

0.90 ⫾ 0.57 1.06 ⫾ 0.42 0.88 ⫾ 0.49 0.70 ⫾ 0.46

1.07 ⫾ 0.59 1.02 ⫾ 0.36 0.38 ⫾ 0.21 0.53 ⫾ 0.35

Cr-B, creatinine before; Cr-A, creatinine after; BUN-B, BUN before; BUN-A, BUN after; Cr Cl-B, creatinine clearances before; Cr Cl-A, creatinine clearances after.

genesis of functional and structural injuries secondary to chronic Tac nephrotoxicity as well as the effects of ACEI and AIIRB on the expression of these cytokines and on the underlying renal damage. MATERIALS AND METHODS A chronic Tac nephrotoxicity model was produced in rats by intraperitoneal (ip) administration of the drug. The study group included 32 male and female Wistar rats, weighing 300 to 350 g, which were fed a low-sodium diet with free access to water. The study animals were divided into four groups of eight: group 1 animals were administered 0.5 mL of saline ip for 28 days and served as healthy controls. Group 2 (Tac) animals received 1 mg/kg of Tac daily for 8 weeks. Group 3 (Tac ⫹ Q) and group 4 (Tac ⫹ V) animals were treated with the same daily dose of Tac for 8 weeks in addition to quinapril (Aquitel, Pfizer, Turkey) 10 mg/kg/d or valsartan (Diovan, Novartis, Turkey) 40 mg/kg/d via the orogastic route, respectively. The animals were housed four per cage and maintained under controlled environmental conditions. All animal procedures were performed in accordance with the Declaration of Helsinki and The Guide for the Care and Use of Laboratory Animals. The study protocol was approved by the local ethics committee. At the end of the study period, 24-hour urine was collected and the animals were sacrificed following collection of blood samples to measure serum creatinine, blood urea nitrogen (BUN), creatinine clearance, and tacrolimus levels. Both kidneys were removed for morphological examination. Tac levels were determined by radioimmunassay using Tacrolimus II ref 3C10 34-3091/R6 IMX System (Abbott Laboratories, USA). Renal tissues were examined for evidence of Tac toxicity, including tubulointerstitial damage and arteriolar hyalinosis. The degree of tubulointerstitial fibrosis was evaluated semiquantitatively in trichrome-stained specimens.22 Hyalinosis of the afferent arterioles was determined by counting the juxtaglomerular arterioles available for examination with a minimum of 50 glomeruli per biopsy assessed in periodic acid-Schiff–stained specimens and

scored semiquantitatively.23 Renal tissues were stained immunohistochemically and evaluated semiquantitatively for TGF-␤ (NCLTGFB; Novocastra), PDGF (NCL-PDGF; Novocastra), IL-6 (M-19; sc-1265; Santa Cruz Biotechnology), VEGF (C-1; sc-7269; Santa Cruz Biotechnology), and BMP-7 (N-19; sc-6899; Santa Cruz Biotechnology). Statistical analyses were performed using student t test to compare pre- and poststudy quantitative variables within the groups, one-way analysis of variance for the analysis of quantitative variables between the groups, and Tukey multiple comparisons if significant. A chi-square test was used to analyse categorical variables. Statistical significance was assumed when P ⬍ .05.

RESULTS

Serum creatinine and BUN levels and creatinine clearances are shown in Table 1. Histological studies revealed significant nephrotoxicity, including increased interstitial mononuclear cell infiltration, focal vacuolization, and calcifications as well as striped fibrosis in the interstitium among G2, G3, and G4 (Table 2). Mean Tac levels were 5.65 ⫾ 2.56 ng/dL for the Tac, 13.36 ⫾ 9.52 ng/dL for the Tac ⫹ Q, and 4.63 ⫾ 1.62 ng/dL for the Tac ⫹ V groups, respectively, a difference that did not reach significance. Immunohistochemical staining revealed increased immune expression of TGF-␤ (P ⬍ .0001), PDGF (P ⬍ .005), and IL-6 (P ⬍ .0001), in G2, G3, G4 compared to G1 (Fig 1). The difference failed to reach statistical significance for VEGF and BMP-7. There was a significantly positive correlation between the severity of interstitial fibrosis, and

Table 2. Mononuclear Cell Infiltration, Focal Vacuolization, and Calcifications Fibrosis*** Groups

MNC (%)*

Vacuolization (%)**

0–0.5

1–1.5

2–2.5

G1 G2 G3 G4

— 75 12.5 14.3

— 62.5 12.5 28.6

7 (100%) — — —

— 4 (50%) 7 (87.5%) 7 (100%)

4 (50%) 1 (12.5%) —

G1, healthy controls; G2, only Tac-treated; G3, Tac ⫹ Quinapril–treated; G4, Tac ⫹ Valsartan treated (group Tac ⫹ V); MNC, mononuclear cell infiltration. *P ⫽ .004. **P ⫽ .04. ***P ⫽ .001.

Fig 1. Cytokine expression in study groups. Cytokine expression was significantly lower in G1 for all cytokine. P ⬍ .001 for TGF G2 versus G3 and G4; P ⬍ .005 for PDGF G2 versus G3 and G4; P ⬍ .001 for IL-6 G2 versus G3 and G4. NS for VEGF. G1, healthy controls; G2, only Tac-treated; G3, Tac ⫹ Quinapril– treated; G4, Tac ⫹ Valsartan–treated (group Tac ⫹ V).

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the severity of hyaline arteriolopathy and TGF-␤ (P ⬍ .001) as well as IL-6 (P ⬍ .05) immune expression. DISCUSSION

We investigated the influence of ACEI and AIIRB on the functional and morphological findings of nephrotoxicity and on the immune expression of TGF-␤, PDGF, VEGF, IL-6, and BMP-7 expression in the glomeruli and interstitium in this rat model of Tac nephrotoxicity. From the functional perspective, Tac nephrotoxicity was only reflected by a slight increase in BUN and creatinine levels, but no other functional impairment was observed in any one of the groups. Despite the lack or paucity of functional impairment, morphological assessment of the kidney specimens revealed marked nephrotoxicity in all groups treated with tacrolimus (G2, G3, G4), although to different degrees. Mononuclear cell infiltration and vacuolization was especially more pronounced in G2, which consisted of rats treated with Tac only. These findings were markedly reduced in groups 3 (ACEI, quinapril) and 4 (AIIRB, valsartan). Furthermore, interstitial fibrosis, which was also present in all the groups treated with Tac, was significantly more prominent in G2 compared with G3 and G4. The same pattern was also observed for hyaline arteriolopathy (Table 3). These findings suggest that the addition of ACEIs or AIIRBs to tacrolimus treatment may have a protective effect for some of the morphological findings of Tac nephrotoxicity. ACEIs may have a relatively better protective effect for hyaline arteriolopathy. These findings also confirmed that functional parameters reflecting renal damage occur much later when some of the morphological changes may have already reached an irreversible stage.24 It has previously been reported that TGF-␤6,7 and PDGF6 have important roles in the pathogenesis of Tac nephrotoxicity. To our knowledge, our study is the first addressing the possible influence of VEGF and IL-6 immune expression in the pathogenesis of Tac nephrotoxicity, in addition to the possible influence of ACEI and AIIRB on the immune expression of the latter cytokines. BMP-7 is an antifibrotic growth factor previously reported to be downregulated in cyclosporine nephrotoxicty and up-regulated with the additional use of ACEI in cyclosporine-induced nephrotoxicity.25 Our results revealed a significantly upregulated expression of TGF-␤, PDGF, VEGF, and IL-6 in all groups treated with Tac compared to the control group. Table 3. Afferent Arteriolopathy Afferent arteriolopathy Groups

None (n, %)

Early Hyalinosis (n, %)

Late Hyalinosis (n, %)

G1 G2 G3 G4

7 (100%) — 1 (12.5) —

— 4 (50%) 7 (87.5) 4 (57.1%)

— 4 (50%) — 3 (42.9%)

P ⫽ .0001. G1, healthy controls; G2, only Tac-treated; G3, Tac ⫹ Quinapril–treated; G4, Tac ⫹ Valsartan–treated (group Tac ⫹ V).

This finding had a significantly positive correlation with the severity of interstitial fibrosis and hyaline arteriolopathy. BMP-7 expression, however, had a tendency to decrease in all groups treated with Tac compared to the control group but this observed difference failed to reach statistical significance. TGF-␤, PDGF, and IL-6 expression was also significantly decreased in groups 3 and 4 compared to G2. BMP-7 expression were similar in all Tac-treated groups and did not seem to be influenced by additional ACEI or AIIRB use in our study. A longer period of drug administration may be needed to see this effect. In conclusion, the results of our study suggested that ACEI and AIIRB may have protective roles in Tac nephrotoxicity. This protective effect may be through downregulation of TGF-␤, PDGF, and IL-6 expression. REFERENCES 1. Ochiai T, Ishibashi M, Fukao K, et al: Japanese multicenter studies of FK 506 in renal transplantation. Japanese FK 506 Study Group. Transplant Proc Feb 27:50, 1995 2. Jurewicz WA: Tacrolimus versus cyclosporin immunosuppression: long-term outcome in renal transplantation. Nephrol Dial Transplant 18(suppl 1):i7, 2003 3. Martin MF: Nephrotoxic effects of immunosuppression. Mayo Clin Proc 69:191, 1994 4. Maes BD, Vanrenterghem Y: Cyclosporine: advantages versus disadvantages vis-à- vis tacrolimus. Transplant Proc 36(suppl 2S):40S, 2004 5. Mihatsch MJ, Kyo M, Morozumi K, et al: The side-effects of ciclosporine-A and tacrolimus. Clin Nephrol 49:356, 1998 6. Jeong HJ, Kim YS, Hong IC: Vascular endothelin, TGF-beta, and PDGF expression in FK506 nephrotoxicity of rats. Transplant Proc 30:3596, 1998 7. Khanna A, Plummer M, Bromberek C, et al: Expression of TGF-beta and fibrogenic genes in transplant recipients with tacrolimus and cyclosporine nephrotoxicity. Kidney Int 62:2257, 2002 8. Egido J: Vasoactive hormones and renal sclerosis. Kidney Int 49:578, 1996 9. Wolf G, Neilson EG: Angiotensin II as a renal growth factor. J Am Soc Nephrol 3:1531, 1993 10. Ruiz-Ortega M, Lorenzo O, Suzuki Y, et al: Proinflammatory actions of angiotensin II. Curr Opin Nephrol Hypertens 10:321, 2001 11. Border WA, Noble NA: Interactions of transforming growth factor-␤ and angiotensin II in renal fibrosis. Hypertension 31:181, 1998 12. Kagami S, Border WA, Miller DE, et al: Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerülar mesengiyal cells. J Clin Invest 93:2431, 1994 13. Branton MH, Kopp JB: TGF-beta and fibrosis. Microbes Infect 1:1349, 1999 14. Zhao Q, Ishibashi M, Hiasa K, et al: Essential role of vascular endothelial growth factor in angiotensin II-induced vascular inflammation and remodeling. Hypertension 44:264, 2004 (Epub 2004 Jul 19) 15. Imanishi T, Hano T, Nishio I: Angiotensin II potentiates vascular endothelial growth factor-induced proliferation and network formation of endothelial progenitor cells. Hypertens Res 27:101, 2004 16. Rizkalla B, Forbes JM, Cooper ME, et al: Increased renal vascular endothelial growth factor and angiopoietins by angiotensin II infusion is mediated by both AT1 and AT2 receptors. J Am Soc Nephrol 14:3061, 2003

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DENIZ, ÖG˘U¨TMEN, ÇAKALAG˘AOG˘LU ET AL 22. Shihab FS, Bennett 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 23. Islam M, Burke JF Jr, McGowan TA, et al: Effect of anti-transforming growth factor-beta antibodies in cyclosporineinduced renal dysfunction. Kidney Int 59:498, 2001 24. Laskow DA, Neylan JF 3rd, Shapiro RS, et al: The role of tacrolimus in adult kidney transplantation: a review. Clin Transplant 12:489, 1998 25. Tug˘lular S, Gogas Yavuz D, Çakalag˘aog˘lu F, et al: Cyclosporine-A induced nephrotoxicity is associated with decreased renal bone morphogenetic protein-7 expression in rats. Transplant Proc 36: 131, 2004