Prevention of Interstitial Fibrosis of Renal Allograft by Angiotensin II Blockade

Prevention of Interstitial Fibrosis of Renal Allograft by Angiotensin II Blockade A. Ishikawa, M. Tanaka, N. Ohta, S. Ozono, and T. Kitamura ABSTRACT We previously confirmed that losartan (LOS), an angiotensin-II (A-II) receptor blocker, diminished plasminogen activator inhibitor-1 (PAI-1) in cyclosporine (CsA)-treated renal graft recipients. Because PAI-1 is known to correlate with tissue fibrosis, we speculated that LOS would have the potential to prevent renal graft interstitial fibrosis. In this study, we focused our attention on the LOS-induced histopathologic changes in renal grafts. Out of 24 CsA-treated normotensive kidney transplanted patients, 8 began to take 25 to 50 mg/day of LOS soon after kidney transplantation (group 1). Eight did so 2 years after kidney transplantation (group 2). Eight received no ARBs as a control group (group 3). PAI-1 levels were monitored every 3 months for 2 years. Renal graft biopsy was performed on all participants, with informed consent, before and 2 years after the onset of this study. The biopsy specimens were stained with periodic acid-methenamine-silver (PAM)-Masson stain for light-microscopic examination. Fibrotic areas in each biopsy specimen were measured using the LUZEX-III image analyzing system. Statistical analysis was performed using Student’s t-test. When we considered the pre-value of PAI-1 in each patient as 100%, the mean percent value of PAI-1 at 2 years after the onset of this study of groups 1, 2, and 3 were 81.5 ⫾ 10.3%, 90.1 ⫾ 12.5%, and 116.8 ⫾ 11.9%, respectively (P ⬍ .01 groups 1 and 2 vs group 3). Light-microscopic examination revealed less remarkable renal interstitial fibrosis among LOS administered groups. A-II blockade may be a key to prevent renal graft interstitial fibrosis.

I

N OUR PREVIOUS clinical study, we confirmed that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator, was diminished by losartan potassium (LOS), an angiotensin-II (A-II) receptor blocker (ARB), among cyclosporine (CsA)-treated renal graft recipients.1 Because PAI-1 is known to correlate with tissue fibrosis,2,3 we speculated that LOS may have the potential to prevent renal graft interstitial fibrosis. In this study, we focused our attention on LOS-induced histopathologic changes, especially the severity of interstitial fibrosis of renal grafts. METHODS We selected 24 CsA-treated normotensive (⬍130/80 mmHg) kidney transplanted patients with no acute rejection episodes. The original renal disease of all patients was chronic glomerulonephritis. CsA, mycophenolate mofetil, and steroids were the immunosuppressants for all participants. Because we selected patients strictly to equalize their historical and demographic backgrounds, no significant differences were seen in duration of hemodialysis, donor age, graft condition, and so on among the three groups.

Among 24 patients, 8 (4 men and 4 women; 49.6 ⫾ 5.6 years old) began 25 to 50 mg/d of LOS soon after kidney transplantation (group 1, early starters); 8 (4 men/4 women, 50.6 ⫾ 6.3 years old) did so at 2 years after kidney transplantation (group 2, late starters); and 8 (4 men/4 women; 47.2 ⫾ 6.4 years old) received no ARBs as a control group (group 3). Plasma PAI-1 level was measured every 3 months for 2 years by a latex photometric immunoassay method.4 With informed consent renal graft biopsies were performed on all participants, before and 2 years after the onset of this study. We

From the Department of Urology, Faculty of Medicine, the University of Tokyo (A.I., T.K.); the Growth Factor Division, National Cancer Center Research Institute (M.T.); the Department of Urology, Yaizu Municipal General Hospital (N.O.); and the Department of Urology, Hamamatsu University School of Medicine (S.O.), Tokyo, Japan. Address reprint requests to Dr Akira Ishikawa, Department of Urology, Faculty of Medicine, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan. E-mail: [email protected]

0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.085

© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

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Transplantation Proceedings, 38, 3498 –3501 (2006)

PREVENTING INTERSTITIAL FIBROSIS

3499 Table 1. Change in Blood Pressure

Group 1 (n ⫽ 8) Group 2 (n ⫽ 8) Group 3 (n ⫽ 8)

Pre

1 Year

2 Years

122.3 ⫾ 6.2/74.8 ⫾ 4.3 122.5 ⫾ 4.4/76.8 ⫾ 2.8 122.3 ⫾ 4.3/77.3 ⫾ 8.4

117.8 ⫾ 6.1/71.5 ⫾ 3.8 120.0 ⫾ 3.3/73.3 ⫾ 3.4 120.0 ⫾ 4.7/73.5 ⫾ 2.1

119.5 ⫾ 5.0/72.3 ⫾ 2.7 119.8 ⫾ 2.3/73.5 ⫾ 2.8 120.5 ⫾ 5.6/73.4 ⫾ 2.9

Table 2. Change in PAI-1 Level Pre (ng/mL)

Group 1 (n ⫽ 8) Group 2 (n ⫽ 8) Group 3 (n ⫽ 8)

RESULTS

1 Year (ng/mL)

2 Years (ng/mL)

19.4 ⫾ 7.3*

15.2 ⫾ 5.8

15.7 ⫾ 6.1

31.6 ⫾ 6.2

29.6 ⫾ 6.5**

28.3 ⫾ 5.3***

20.9 ⫾ 6.3*

22.8 ⫾ 6.8**

24.3 ⫾ 7.0***

These data are “original” plasma PAI-1 values, which were measured by latex photometric immunoassay method. *P ⬍ .01 vs. group 2. **P ⬍ .03 vs. group 1. ***P ⬍ .02 vs. group 1.

Table 3. Change in Percent PAI-1 Level

Group 1 (n ⫽ 8) Group 2 (n ⫽ 8) Group 3 (n ⫽ 8)

Pre (%)

1 Year (%)

2 Years (%)

100.0

78.6 ⫾ 7.5*

81.5 ⫾ 10.3*

100.0

93.4 ⫾ 10.0*

90.1 ⫾ 12.5*

110.0 ⫾ 10.5

100.0

116.8 ⫾ 11.9

*P ⬍ 0.01 vs. group 3.

set the interval for biopsy at 2 years; however, there was no special reason for that. We just thought that 2 years might be enough to evaluate changes. The biopsy specimens were processed and stained with PAM-Masson (PM) for the light-microscopic examinations. Fibrotic areas in each PM-stained specimen were measured using the LUZEX III image analyzing system (NIRECO Co., Tokyo, Japan) and compared among the three groups. PAI-1 expression was also examined with homogenized biopsy cores by a Western blotting method using a monoclonal antibody against human PAI-1 (American Diagnostica Inc, #379, Conn). Although the number of cases was small, statistical analysis was performed using Student t-test.

100 80

The blood pressure of each patient was well controlled at 110 to 130/60 to 70 mmHg and neither hyper- nor hypotensive episodes were observed during the study (Table 1). No acute rejection episodes occurred in those patients for 2 years. Changes in PAI-1 levels are summarized in Table 2 To eliminate the difference among individuals, we translated the “ng/mL” value of PAI-1 into “percent” value, considering the pre-value of PAI-1 in each patient as 100%. The mean percent values of PAI-1 at 1 year (2 years) after the onset of the study in groups 1, 2, and 3 were 78.6 ⫾ 7.5 (81.5 ⫾ 10.3), 93.4 ⫾ 10.0 (90.1 ⫾ 12.5), and 110.0 ⫾ 10.5 (116.8 ⫾ 11.9) percent, respectively (Table 3). Statistically significant differences were seen between plasma PAI-1 levels of groups 1 and 2 and group 3 (P ⬍ .01). There may be a dose dependence of PAI-1 suppression by LOS (P ⬍ .1 at 1 year; Fig 1). PAI-1 expression was suppressed in LOS administered patients (Fig 2). Serum creatinine (S-Cr) levels of LOS administered groups were increased slightly but temporarily (Table 4). Light-microscopic examinations with PM-stained samples (Fig 3 and 4) and the LUZEX III image analyzing system revealed that renal interstitial fibrosis was less remarkable among samples from LOS-administered groups (Table 5). DISCUSSIONS

Our study suggested that LOS may diminish plasma PAI-1 levels and prevent renal graft interstitial fibrosis without significantly reducing systemic blood pressure. Diez5 pointed out that LOS may possess a number of properties independent of its antihypertensive effects. Mehta et al6 reported that A-II stimulated both expression and release of PAI-1. Huang et al7 demonstrated that

NS

P < 0.1 80.4±6.6 (n=5)

75.6±7.2 (n=3)

82.2±7.6 (n=5)

80.1±7.8 (n=3)

60 40 20 0

1y

2y

Fig 1. Dose dependency on the PAI-1 suppression effect of LOS? Out of eight patients in group 1 (ie, early starters), five received 25 mg/d and three received 50 mg/d of LOS. Although the number of cases was very small, there might be some dose dependency for PAI-1 inhibition effect of LOS.

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ISHIKAWA, TANAKA, OHTA ET AL

Fig 2. PAI-1 expression examined by Western blot using ADI #379.

rennin-induced transforming growth factor beta-1 stimulated PAI-1 production. Yoshida et al8 indicated that interleukin-1 beta increased A-II–induced PAI-1 secretion in astrocytes through A-II type 1 receptor induction. These reports suggested that many factors are concerned in PAI-1 expression. Therefore, further studies are needed; however, based on the data obtained from the present study on a small number of strictly selected patients, we believe that A-II blockade may be a key for PAI-1 reduction, and PAI-1 reduction itself may be one of properties of LOS. Matsuo et al3 reported that PAI-1 was directly involved in interstitial fibrosis. Therefore, we supposed that PAI-1 reduction by LOS would play an important role in prevention of renal graft interstitial fibrosis. This antifibrotic effect Table 4. Change of S-Cr Level

Group 1 (n ⫽ 8) Group 2 (n ⫽ 8) Group 3 (n ⫽ 8)

Pre (mg/dL)

3 Months (mg/dL)

1 Year (mg/dL)

2 Years (mg/dL)

1.1 ⫾ 0.3*

1.4 ⫾ 0.1**

1.2 ⫾ 0.2***

1.3 ⫾ 0.3****

1.8 ⫾ 0.5

2.1 ⫾ 0.7**

2.0 ⫾ 0.6

2.0 ⫾ 0.7

1.1 ⫾ 0.2*

1.1 ⫾ 0.2

1.2 ⫾ 0.2***

1.3 ⫾ 0.2****

*P ⬍ 0.01 vs. group 2. **P ⬍ .001 vs. group 3. ***P ⬍ .04 vs. group 2. ****P ⬍ .02 vs. group 2.

Fig 3. PAM stained renal tissue: a case from group 1. Left, pre (s-Cr 1.3 mg/dL/right, post (S-Cr 1.0 mg/dL).

of LOS would be expected not only on “early” but also on “late” starters, as shown in group 2. Although the existing fibrosis could not disappear, LOS prevented worsening in renal graft tissue. PAI-1 seems likely to increase gradually after CsAtreated renal transplantation, as was shown in group 3 (the group not administered LOS). The initial PAI-1 level of group 2 was also significantly higher than those of groups 1 and 3. We think that CsA stimulates the local (intrarenal) rennin–angiotensin–aldosterone (RAA) axis).9 CsA activation of the RAA axis causes A-II (upregulate) PAI-1 expression, inducing renal graft interstitial fibrosis. Thus, we suppose that A-II blockade may be important for all CsA-treated renal allograft recipients. As far as our present study showed, 25 to 50 mg/d of LOS was sufficient to prevent fibrosis with no serious effects on blood pressure. Interstitial fibrosis is one histopathologic feature of chronic allograft nephropathy,10 a major cause of renal graft loss. Control of blood pressure itself might be significant; however, based on the results of our present study, A-II blockade or PAI-1 reduction may prolong renal allograft survival. For this study, we selected patients with the same historical and demographic backgrounds; however, we know that the number of cases is rather small. It is necessary for large-scale trial, allowing important factors to be evaluated in multivariate fashion.

PREVENTING INTERSTITIAL FIBROSIS

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Fig 4. PAM stained renal tissue: a case from group 3. Left, Pre (s-Cr 1.0 mg/dL)/right, post (S-Cr 1.3 mg/dL). Table 5. Change of the Rate of Fibrotic Area Pre (%)

Group 1 (n ⫽ 8) Group 2 (n ⫽ 8) Group 3 (n ⫽ 8)

4.6 ⫾ 7.6 13.8 ⫾ 10.2*** 5.1 ⫾ 8.6

Post (%)

5.8 ⫾ 10.6* 16.2 ⫾ 14.8 11.2 ⫾ 9.4**

*P ⬍ .05 vs. group 3. **P ⬍ .05 vs. pre-value of group 3. ***P ⬍ .05 vs. groups 1 and 3.

In conclusion, LOS diminished PAI-1. A-II blockade and/or PAI-1 reduction may be a key to prevent renal interstitial fibrosis, and contribute to extended renal allograft survival.8 REFERENCES 1. Ishikawa A, Ohta N, Ozono S, et al: Inhibition of plasminogen activator inhibitor-1 by angiotensin II receptor blockers on cyclosporine-treated renal allograft recipients. Transplant Proc 37:994, 2005 2. Revelo MP, Federspiel C, Helderman H, et al: Chronic allograft nephropathy: expression and localization on PAI-1 and PPAR-gamma. Nephrol Dial Transplant 20:2812, 2005

3. Matsuo S, Lopez-Guisa JM, Cai X, et al: Multifunctionality of PAI-1 in fibrogenesis: evidence from obstructive nephropathy in PAI-1-overexpressing mice. Kidney Int 67:2221, 2005 4. Ono T, Sogabe N, Ogura M, et al: Autmated latex photometric immunoassay for total plasminogen activator inhibitor-1 in plasma. Clin Chem 49:987, 2003 5. Diez J: Review of the molecular pharmacology of Losartan and its possible relevance to stroke prevention in patients with hypertension. Clin Ther 28:832, 2006 6. Mehta JL, Li DY, Yang H, et al: Angiotensin II and IV stimulate expression and release of plasminogen activator inhibitor-1 in cultured human coronary artery endothelial cells. J Cardiovasc Pharmacol 39:789, 2002 7. Huang Y, Wongamomtham S, Kasting J, et al: Renin increases mesangial cell transforming growth factor-beta 1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms. Kidney Int 69:105, 2006 8. Yoshida H, Imaizumi T, Tanji K, et al: Interleukin-1 beta enhances the angiotensin-induced expression of plasminogen activator inhibitor-1 through angiotensin receptor upregulation in human astrocytes. Brain Res 38:1073, 2006 9. Ishikawa A, Sakaguchi M, Nakano M, et al: Cyclosporine induced nephrotoxicity and juxtaglomerular apparatus in mice. Nippon Hinyokika Gakkai Zasshi 82:1286, 1991 10. Nankivell BJ, Chapman JR: Chronic allograft nephropathy: current concepts and future directions. Transplantation 81:643, 2006