EFFECTS OF NITRIC OXIDE SYNTHESIS INHIBITION ON FK506-INDUCED NEPHROTOXICITY IN RATS

Abstract

This study was designed to evaluate the role of nitric oxide (NO) in FK506-induced nephrotoxicity by administering an inhibitor of NO synthesis, Nω-nitro-L-arginine methyl ester (L-NAME) to rats treated with FK506. After one week of treatment with FK506 (3.2 mg/kg/day, intramuscularly) and/or L-NAME (5 mg/100 mL of L-NAME in the drinking water), the arterial pressure, urinary NOx, and parameters for renal function were measured, and histological analysis of the kidney was made. In the L-NAME without FK506 group, L-NAME administration effectively inhibited urinary NOx excretion and increased mean arterial pressure (MAP) without any change in renal function. In the FK506 without L-NAME group, FK506 treatment showed increase in urinary NOx excretion and mild renal dysfunction. In the FK506 with L-NAME group, urinary NOx excretion was decreased by L-NAME administration and renal function was significantly worsened than FK506 without L-NAME group. The plasma creatinine, BUN and urinary N-acetyl-β-D-glucosaminidase increased 2-, 3-, and 3-fold, respectively and the creatinine clearance was reduced by 50% as compared with that in the FK506 without L-NAME group. Histological analysis revealed severe interstitial fibrosis and tubular atrophy in the FK506 + L-NAME treatment group. Thus, results suggest that NO synthesis is enhanced in the kidney during FK506-induced nephrotoxicity and that NO synthesis inhibition aggravates FK506-induced nephrotoxicity. NO may play a protective role attributable to the balance of vasoactive substances in FK506-induced nephrotoxicity.

• FK506
• Nephrotoxicity
• Nitric oxide
• Nω-nitro-L-arginine methyl ester

INTRODUCTION

FK506 is a potent immunosuppressive agent produced as a fermentation product of Streptomyces tsukubaensis, which possesses 100 times more potent immunosuppressive properies than CsA in vitro [1-2]. FK506 has been widely used in the prophylaxis of allograft rejection and rescue of patients with refractory rejection after human liver, kidney, and heart transplantation not responding to either CsA or OKT3 because of its indubitable efficacy in the prevention of organ allograft rejection [3-6]. However, nephrotoxicity is a serious complication of FK506 therapy and limits its clinical use [7-9]. Although little is known about underlying mechanism of FK506 nephrotoxicity, some studies have suggested that FK506 nephrotoxicity is characterized by disturbances of renal hemodynamics that are attributed to an imbalance in the release of vasoactive substances. On the one hand, an increased release of vasoconstricting factors, such as thromboxane [[10]], [[13]], endothelin [11-12], and angiotensin II [[13]], and, on the other, a decrease in vasodilating factors such as prostacyclin [13-14]. Nitric oxide (NO) is synthesized from the terminal guanidino nitrogen atom(s) of L-arginine by nitric oxide synthase and is a vasoactive factor that plays a key role in the control of the renal vascular tone and blood flow [[16]]. Involvement of NO in the FK506-induced nephrotoxicity, however, has not been well defined. This study was designed to evaluate the role of NO in FK506-induced nephrotoxicity by administering an inhibitor of NO synthesis, Nω-nitro-L-arginine methyl ester (L-NAME) to rats treated with FK506.

MATERIALS AND METHODS

Drugs and Methods of Administration

FK506 (Fujisawa Pharmaceutical Co. Ltd. Osaka, Japan) was disssolved in physiological saline to a final concentration of 8 mg/mL and was administered intramuscularly (i.m.) at 3.2 mg/kg body weight, once a day. L-NAME and D-NAME (Sigma Chemical Co. St. Louis, USA) was dissolved in drinking water at a concentration of 5 mg/100 mL. Rats were allowed free access to tap L-NAME water and average dose of L-NAME taken was 0.58 ± 0.31 mg per day per rat. Pentobarbital was purchased from Abbott Lab. (North Chicago, U.S.A.)

Experimental Design

Male wistar rats weighing 240–260 g, age 11 weeks, were housed in individual cages in a temperature- and light-controlled environment. The weight-matched rats were randomized into four groups, each of which consisted of six rats:

1. Control group: Rats received a daily i.m. injection of 0.1 mL physiological saline.

2. L-NAME group: Rats received a daily i.m. injection of 0.1 mL physiological saline and L-NAME drinking water administration. In addition to six rats, D-LNAME was used instead of L-NAME.

3. FK506 group: Rats received a daily i.m. injection of FK506 (3.2 mg per kg/day).

4. FK506+L-NAME group: Rats received a daily i.m. injection of FK506 (3.2 mg/kg/day) and L-NAME drinking water administration. In addition to six rats, D-LNAME was used instead of L-NAME.

After one week of the treatment, the tail blood pressure was measured with a plethysmograph (Ueda Co. UR-1000, Tokyo, Japan) and the rats were placed in metabolic cages for a 24-hour urine collection. Then the rats were anesthetized with pentobarbital administered intraperitoneally (5 mg per 100 g body weight), the blood sample was collected from the abdominal aorta, and the kidneys were fixed by the intravascular perfusion of 4% formalin and 1% glutaraldehyde in phosphate buffer solution, pH 7.4. The total osmolality of the buffer solution was 200 mosmol/L [[17]]. The perfusion was performed for 5 minutes under the pressure of 140–160 mmHg by using a pump as previously reported [[18]].

Analytical Methods

Urinary and plasma sodium and potassium were determined with a flame photometer (Hitachi 710, Hitachi Co., Tokyo, Japan). Urinary and plasma creatinine (Cr) and magnesium, BUN, and urinary N-acetyl-β-D-glucosaminidase (NAG) were determined with an autoanalyzer (Hitachi 7170, Hitachi Co, Tokyo, Japan), 24 hours' clearance of creatinine (CCr), 24 hours' urinary excretion of sodium (UENa), potassium (UEK), and magnesium (UEMg) were calculated by standard formula. Urinary NOx (nitrate; NO3 + nitrite; NO2) assay was done by the NO analysis system ENO-200 (Eicom Co. Lit., Kyoto, Japan) in the Griess reaction [[20]].

Histological Analysis

Multiple longitudinal sections of the kidneys, were processed and embedded in paraffin. 2 to 4 μm sections were stained with hematoxylin and eosin (H&E), and periodic acid-Schiff (PAS). The histological findings of tubular and interstitial changes were graded by semiquantitative analysis in a blinded fashion by one observer (S.R.). Interstitial fibrosis score (IFS) was semiquantitatively analyzed according to the following criteria [[21]]: 0: normal interstitium and tubules; 1: mild fibrosis with minimal interstitial thickening between the tubules; 2: modest fibrosis with moderate interstitial thickening between the tubules, and 3: severe fibrosis with severe interstitial thickening between the tubules. Tubular injury was defined as presence of tubular dilatation, tubular atrophy, tubular cast formation, sloughing of tubular epithelial cells, and/or thickening of the tubular basement membrane. Proximal tubular injury score (PTS) was semiquantitatively analyzed according to the following criteria [[22]]: 0: no tubular injury; 0.5: <5% of tubules injured; 1: 5–20% of tubules injured; 1.5: 21–35% of tubules injured; 2: 36–50% of tubules injured; 2.5: 51–65% of tubules injured, and 3: >65% of tubules injured.

Statistical Analysis

Results are presented as the mean ± SD, and all statistical analyses were calculated with Statview for Macintosh 5.0 (SAS Institute Inc. U.S.A.). The level of statistical significance was chosen as p < 0.05.

RESULTS

1. Parameters for renal function and urinary NOx excretion. The results of physiologic data, renal function and urinary NOx excretion are summarized in Table 1. Weight gain was observed in the control group (275.3 ± 2.04 g posttreatment vs. 251.2 ± 8.3 g pretreatment, p < 0.05) and L-NAME group (274 ± 3.35 g posttreatment vs. 253.7 ± 8.6 g pretreatment, p < 0.05). The body weight in the FK506-treatment group was not significantly changed (243.5 ± 5.4 g posttreatment vs. 253.7 ± 8.6 g pretreatment, N.S.), but the body weight of FK506+L-NAME group was markedly decreased (214.3 ± 7.5 g posttreatment vs. 251.7 ± 3.1 g pretreatment, p < 0.05). One week of treatment with L-NAME decreased urinary NOx excretion in the L-NAME group (9.49 ± 3.75 μmol/day in L-NAME group vs. 24.19 ± 4.2 μmol/day in control group, p < 0.05) and induced a high mean arterial pressure (MAP) (130.4 ± 2.8 mmHg in L-NAME group vs. 114.8 ± 1.8 mmHg in control group, p < 0.05). However, there was no change in the renal function (Tab. 1). One week of treatment with FK506 induced a mild decrease in CCr and an increase in BUN, as well as decrease in plasma Mg (Tab. 1). The 24–hours urinary NOx excretion in FK506 group was significantly higher than that in control group (46.45 ± 6.87 μmol/day in FK506 group vs. 24.19 ± 4.2 μ mol/day in control group, p < 0.05). In comparison with FK506 group, lower urinary NOx excretion in the FK506+L-NAME group was seen (9.76 ± 3.52 μmol/day in FK506+L-NAME group vs. 46.45 ± 6.87 μ mol/day in FK506 group, p < 0.05), suggesting that L-NAME administration inhibited NO production. Meanwhile, the rats treated with both FK506 and L-NAME showed a more severe impairment of renal function than the rats treated with FK506 alone. Plasma Cr, BUN, and the urinary NAG excretion in the FK506+L-NAME group were 2-, 3-, and 3-fold higher than those in the FK506 group respectively, and creatinine clearance was reduced to 50% as compared with that in the FK506 group (Tab. 1). D-NAME did not show any effect on renal function and urinary NOx excretion in rats.

2. Histological findings. Histological findings were shown in Figure 1. In rats treated with only FK506, mild interstitial fibrosis and tubular atrophy were seen in the renal cortex. In contrast, when both FK506 and L-NAME were given, the interstitial fibrosis and tubular atrophy were much more extensive (Fig. 1). Semiquantitative analysis showed that there was a statistical significance in PTS and IFS between FK506 group and FK506+L-NAME group (Fig. 2).

Table 1. Physiological Data, Renal Function, and Urinary NOx Excretion in the Rats Receiving One-Week Treatment with FK506 and/or L-NAME

	Control	L-NAME	FK506	FK506 + L-NAME
BW (g)	275.3 ± 2.04	274 ± 3.35	243.5 ± 2.21^a	214.3 ± 7.51-3^a,b,c
MAP (mmHg)	114.8 ± 1.8	130.4 ± 2.81^a	117.8 ± 6	119 ± 3.8
UF (uL/min)	5.5 ± 0.7	5.6 ± 0.9	4.3 ± 0.3	2.7 ± 0.51^a
PNa (mg/dL)	146.2 ± 0.6	145.2 ± 0.6	145.5 ± 0.4	145.1 ± 1.5
PK (mg/dL)	5.7 ± 0.56	6.9 ± 1.1	5.7 ± 0.47	5.2 ± 0.74
PMg (mg/dL)	2.32 ± 0.12	2.55 ± 0.18	1.9 ± 0.11-2^a b	2.56 ± 0.273^c
UENa (uEq/day)	250.5 ± 50.3	327.6 ± 95	314.7 ± 53.3	164 ± 43.3
UEK (uEq/day)	1115 ± 180	1402.3 ± 202.6	1138 ± 293.1	521 ± 1651-3^a,b,c
UEMg (uEq/day)	1.78 ± 0.68	1.36 ± 0.4	1.4 ± 0.28	0.8 ± 0.12
UENAG (U/gCr)	3.68 ± 0.8	5.36 ± 1.77	3.61 ± 0.88	11.75 ± 2.951, 3^a,c
BUN (mg/dL)	18.51 ± 2.29	21.45 ± 1.72	33.11 ± 2.761-2^a,b	117 ± 34.91-2^a,b,c
Cr (mg/dL)	0.33 ± 0.04	0.38 ± 0.04	0.43 ± 0.03	0.99 ± 0.21-3^a,b,c
CCr (mL/100 g/min)	0.62 ± 0.11	0.51 ± 0.07	0.36 ± 0.021^a	0.16 ± 0.041-3^a,b,c
UENOx (umol/day)	24.19 ± 4.2	9.49 ± 3.751^a	46.45 ± 6.781-2^a,b	9.76 ± 3.521, 3^a,c

Note: Control, control rats; L-NAME, rats received L-NAME treatment (5 mg/100 mL in drinking water); FK506, rats received FK506 treatment (3.2 mg/kg/day i.m.); FK506+L-NAME, rats received both FK506 treatment and L-NAME treatment. BW, body weight; MAP, mean arterial pressure; UF, urine flow; PNa, plasma sodium concentration; PK, plasma potassium concentration; PMg, plasma magnesium concentration; UENa, 24 hours' urinary excretion of sodium; UEK, 24 hours' urinary excretion of potassium; UEMg, 24 hours' urinary excretion of magnesium; UENAG, 24 hours' urinary excretion of N-acety-β-glucosaminidase; UENOx, 24 hours' urinary excretion of NO2 + NO3.

^ap < 0.05 vs. control group;

^bp < 0.05 vs. L-NAME group;

^cp < 0.05 vs. FK506 group.

Figure 1. Renal histological changes in the rats receiving one week treatment with FK506 and/or L-NAME (hematoxylin and eosin staining, ×200). 1, control rats; 2, rats received L-NAME treatment (5 mg/100 mL in drinking water); 3, rats received FK506 treatment (3.2 mg/kg/day i.m.); 4, rats received both FK506 treatment and L-NAME treatment. In rats treated with only FK506, mild interstitial fibrosis and tubular atrophy are seen in the renal cortex. In contrast, when both FK506 and L-NAME are given, the interstitial fibrosis and tubular atrophy are much more extensive.

Figure 2. Semiquantitative analysis of histological changes of the kidney in the rats received one week treatment with FK506 and/or L-NAME. Control: control rats. L-NAME: rats received L-NAME treatment (5 mg/100 mL in drinking water). FK506: rats received FK506 treatment (3.2 mg/kg/day i.m.). FK506 + L-NAME: rats received both FK506 treatment and L-NAME treatment. PTS: proximal tubular injury score. IFS: interstitial fibrosis score. a: p < 0.05 vs. control group. b: p < 0.05 vs. L-NAME group. c: p < 0.05 vs. FK-506 group.

DISCUSSION

Baylis et al. have reported that two months treatment with the dose of L-NAME (5 mg/100 mL in drinking water) was able to induce hypertension and decrease of GFR in the rats [[19]]. In our study, the L-NAME group was designed to demonstrate that the dose of L-NAME administration for one week was enough to inhibit NO synthesis. We found that treatment with this dose of L-NAME for one week in conscious control rats reduced the urinary NOx excretion and induced a significant increase in the MAP, thus suggesting that this treatment is effective in producing a systemic and renal NO blockade in rats. However, one-week treatment did not induce renal dysfunction and morphological changes in our study. Probably one week administration of L-NAME is not enough to induce changes in renal function and morphology.

Many studies hitherto conducted that FK506 can induce a dose-dependent renal dysfunction with tubular and interstitial changes in rat kidneys [[18]], [21-22]. In our study, one-week treatment of FK506 induced a slight decrease in GFR and a slight increase in BUN. These changes were associated with minimal morphological alteration—mild interstitial fibrosis and tubular atrophy in renal cortex. These results revealed the limited nephrotoxicity of one week treatment of FK506.

The mechanism of FK506-induced nephrotoxicity is not clear. Available data suggested that FK506-induced nephrotoxicity is attributed to an imbalance in the release of vasoactive substances, that is, an increased release of vasoconstricting factors and a decrease in vasodilating factors [11-15]. NO is produced from L-arginine by the action of NOS in the kidney and is a vasoactive factor that plays a key role in maintaining the vascular tone. However, involvement of NO in the FK506-induced nephrotoxicity has not been well defined. In our study, a high urinary NOx excretion was found in rats treated by FK506 in comparson with control group (Tab. 1). NOx (NO2 + NO3) is the stable end product of NO, and NO synthesis can be indirectly evaluated by measuring the NOx. So this suggested that NO synthesis is enhanced by the FK506 treatment in the kidney. The mechanism of increased NO synthesis is not clear. It may be secondary to an increase of vasoconstricting factors [10-13], and the same mechanism has been suggested in the cyclosporineA-induced nephrotoxicity [[23]]. In contrast with a high urinary NOx excretion in the FK506 group, a low urinary NOx excretion in the FK506+L-NAME group was seen, suggesting that L-NAME administration inhibited the increased NO production that was induced by FK506 treatment. Meanwhile, the rats treated with FK506 and L-NAME in combination showed severe renal dysfunction. As Table 1 shows, plasma Cr, BUN and the urinary NAG excretion in the FK5064+L-NAME group were increased 2-, 3-, and 3-fold, respectively and CCr was reduced by 50% as compared with that in the FK506 group. In agreement with the severe renal dysfunction, extensive interstitial fibrosis and tubular atrophy were also seen in the renal cortex in the rats treated with both FK506 and L-NAME (Fig. 1 and Fig. 2). So our data suggest that NO synthesis inhibition aggravates the FK506-induced nephrotoxicity and that NO, a potent vasorelaxing factor produced by endothlium, may play a protective role contributing to the maintenance of balance of vasoactive substances in the FK506-induced nephrotoxicity. However, the mechanism that NO synthesis is enhanced in the kidney during FK506-induced nephrotoxicity must be determined in future studies.

ACKNOWLEDGMENTS

We thank Fujisawa Pharmaceutical Company for kindly supplying FK506. We also thank Dr. Tetsu Yamana (Department of pathobiology, Yamanashi Medical University, Japan) for the assistance with histological study. This study was supported in part the Grant-In-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No:11470334) to M. Takeda.