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*
Department of Surgery, Division of Immunology and Organ Transplantation,
The University of Texas Medical School at Houston, Houston, Texas
Department of Pathology and Laboratory Medicine, The University of Texas
Medical School at Houston, Houston, Texas
Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering
Cancer Center, New York, New York.
Correspondence to Dr. Barry D. Kahan, Division of Immunology and Organ Transplantation, The University of Texas Medical School at Houston, 6431 Fannin, Suite 6.240, Houston, TX 77030. Phone: 713-500-7400; Fax: 713-500-0785; E-mail: bkahan{at}orgtx71.med.uth.tmc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the exact mechanisms of CsA-induced nephrotoxicity are not understood fully, important components include increased vascular resistance, which produces decreased renal blood flow (10,11); generation of reactive free radicals, which causes both oxidative stress (12) and cytochrome P450 activation (13); upregulated expression of the profibrogenic principle transforming growth factor-ß (14); increased generation and responses of smooth muscle cell calcium to vasoconstrictive stimuli (15); upregulated synthesis and expression of angiotensin II receptors (15); and depressed nitric oxide production by both endothelial and inducible nitric oxide synthases (16). Furthermore, CsA has been reported to promote Fas-mediated (17) apoptosis of LLC-PK1-cultured renal tubular cells in vitro, an effect that is blocked by peptide inhibitors of caspases 3, 8, and 9 (18). Thus, increased vasoconstriction and apoptosis characterize CsA nephrotoxicity.
Because the synergistic immunosuppressive interactions between CsA and RAPA were first documented in rats (19,20), this species represented a logical model to investigate the impact of the drug combination on the kidney. The salt-depleted rat model has been reported to produce functional and histopathologic effects that resemble the dose-dependent impairment of renal function observed in CsA-treated kidney transplant patients (1,10,21,22,23). In contrast, administration of therapeutic doses of RAPA (0.04 to 0.8 mg/kg per d intravenously) caused no significant renal changes in rats (24). At 0.8 mg/kg per d RAPA, normal rats displayed only a marginal elevation in serum creatinine (SCr) values, and spontaneously hypertensive rats showed neither accelerated necrotizing vasculopathy nor tubular atrophy (25). Only supratherapeutic (1.6 to 6.4 mg/kg per d) RAPA doses produced transmural fibrinoid necrosis of vessels in the gastrointestinal submucosa and in the kidney, as well as juxtaglomerular hypertrophy, tubular dilation, basement membrane thickening, vacuolization, and atrophy (25).
Owing to its use in combination with CNA, there is considerable interest in understanding the impact of RAPA on CsA-induced nephrotoxic injuries. Andoh et al. (22) reported that subcutaneous coadministration of RAPA potentiated CsA-induced nephrotoxicity in salt-depleted rats and postulated that the effect was due to enhanced hyperglycemia. In contrast, we suggested that increased drug concentrations contribute to the adverse renal effects displayed by the CsA/RAPA combination, because we had documented elsewhere that a portion of the synergistic effect was due to pharmacokinetic interactions (26), particularly after oral coadministration (27). The present study revealed a predominant role of pharmacokinetic interactions to produce toxic renal exposures of CsA and of dynamic effects to potentiate the lipid as well as the myelosuppressive toxicities.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Drugs
Commercial oral formulations of CsA (Sandimmune; Novartis Research, East
Hanover, NJ) and RAPA (Rapamune; Wyeth-Ayerst, Princeton, NJ) were
administered by oral gavage in a constant volume of 0.2 ml daily for 14 d.
Experimental Groups
After a 7-d conditioning period on low-salt chow, groups of six rats were
assigned randomly to treatment for 14 d with CsA alone (2.5, 5.0, 7.5, 10.0,
15.0, or 20.0 mg/kg per d), RAPA alone (0.4, 0.8, 1.2, 1.6, 3.2, or 6.4 mg/kg
per d), or CsA/RAPA combinations at a fixed 6.25:1 ratio (2.5/0.4, 5.0/0.8,
7.5/1.2, 10.0/1.6, 15.0/3.2, or 20.0/6.4 mg/kg per d), which had been shown to
be the optimal ratio to document synergistic immunosuppression, or at varying
ratios of fixed 5.0 or 10.0 mg/kg per d CsA doses with ascending amounts of
RAPA (0.4, 0.8, 1.2, 1.6, 3.2, or 6.4 mg/kg per d). In addition, there were
two untreated control groups, each composed of six rats: one fed a low-salt
diet and the other fed a normal diet.
After receiving the final drug doses on day 14, the animals were placed in metabolic cages for 24-h urine collections and GFR measurements, by use of iohexol in the Renalyzer PRX 90 (Provalid AB, Lund, Sweden). Inman et al. (28) documented in rats that this method provides an accurate and reliable measure of GFR, compared with inulin determinations. In addition, urinary sodium, potassium, magnesium, calcium, phosphate, and creatinine levels were quantified by use of established methods in our clinical chemistry laboratory. Upon completion of the urine collection, the animals were anesthetized with intraperitoneal pentobarbital (Abbott, Chicago, IL) to obtain whole-blood samples for complete blood counts, CsA and RAPA concentration measurements, and serum aliquots for sodium, potassium, magnesium, calcium, phosphate, uric acid, cholesterol, creatinine, and lipoproteins. The laboratory results, presented as mean values ± deviants, were compared for statistical significance by use of ANOVA; P < 0.05 was accepted as significant.
The left kidney was removed and split in half. One half of the left kidney and a 2-g sample of the right lateral lobe of the liver were used for drug concentration measurements. The other half of the left kidney was fixed in buffered 10% formalin and processed overnight; 3-µm histologic sections were stained with progressive hematoxylin-eosin, periodic acid-Schiff, or Masson's Trichrome reagents. Two independent pathologists (J.C. and R.V.), who were blinded to treatment assignments, used semiquantitative scales of light microscopic criteria to assess the degree of vasculopathy, glomerular changes, and tubulointerstitial damage in multiple kidney sections. Tubular and glomerular changes were graded separately as follows: 0, no changes; 1+, <5%; 2+, 5 to 25%; 3+, 26 to 50%; and 4+, >50% involvement. A similar vascular scale included the following: 0, none; 1+, minimal; 2+, mild; 3+, moderate; and 4+, severe. Although the scores generally were concordant, when they were disparate, a mean value was chosen as the histopathologic grade.
Renal Function
GFR was measured by the iohexol method
(29). The femoral vein and
artery, as well as the transplant ureter, were cannulated individually by use
of 10-0 silicone tubing (Baxter, Deerfield, IL). BP, heart rate, and urine
output were monitored with the use of a Micro-Med apparatus (Louisville, KY)
and analyzed with the use of a DMSI 2004 computer program (Micro-Med). BP was
recorded automatically every 30 s and urine output every 5 min. A loading dose
of 1000 mg/kg iohexol (Omnipaque, 300 mg/ml; Nycomed, Inc., Princeton, NJ) was
administered intravenously over 5 min, followed by infusion of 600 mg/kg over
90 min, as recommended by Inman et al.
(28). Urine samples, collected
at 20-min intervals after completion of the loading dose, were analyzed for
iohexol concentrations. Whole-blood samples were obtained at the midpoints of
the urine collections. GFR values (ml/min) were calculated by the formula (U
x V)/P, where U is urinary iohexol concentration (mg/ml), V is urine
output (ml/24 h), and P is plasma iohexol concentration (mg/ml). The results
were presented as mean ± deviants, and statistical significance was
assessed by t test.
Bone Marrow Cellularity
The right femur was harvested, fixed in 10% buffered formalin, decalcified
in formic acid for approximately 1 wk, sectioned (3 to 5 µm), and stained
with hematoxylin and eosin by use of standard techniques. Hematopoiesis was
estimated as the percentage of the marrow space occupied by cellular as
opposed to adipose tissue elements. The average number of megakaryocytes in
four high-power fields (40x) was used to estimate the effects of RAPA
with or without concomitant administration of CsA on platelet formation.
Drug Concentration Measurements
For CsA measurements, whole-blood samples (0.5 ml) were collected into
ethylenediaminetetraacetate-containing tubes (Becton Dickinson, Mountain View,
CA); 1- to 2-g aliquots of hepatic and renal tissues were disrupted by use of
an Ultrasound Homogenizer (Fisher Scientific, Pittsburgh, PA). CsA
determinations were performed by use of an automated fluorescence polarization
immunoassay (TDx; Abbott, Chicago, IL). In contrast to human specimens and on
the basis of supporting data of others
(30,31,32,33,34,35),
we documented elsewhere that the TDx technology provides results similar to
HPLC because rats do not produce CsA metabolites that cross-react
significantly in the TDx assay
(36). CsA concentrations were
expressed as ng/ml for whole blood or ng/g for wet-tissue weight. The
intra-/interassay coefficients of variation for blood and tissue CsA
measurements were 3.2% at 150 ng/ml and 1.7% at 800 ng/ml, and 2.3% at 150
ng/ml and 1.6% at 800 ng/ml, respectively (Napoli KL, Kahan BD, unpublished
observations).
RAPA concentrations were estimated by use of our published method of HPLC with ultraviolet detection (37). Owing to the photosensitivity of RAPA, left kidney and liver samples (1 to 2 g) had to be protected from light during ultrasonic disruption. Briefly, 1 ml of 0.1 M sodium carbonate and 20 ml of methanolic-estradiol-3-methylether, an internal standard, were added to 1 ml of whole blood. After double extraction with 10 ml of t-butyl methyl ether, the pooled supernates were evaporated, reconstituted twice with 150 µl of absolute ethanol, and finally suspended in 100 µl of mobile phase buffer composed of an 85:15 ratio of methanol/water. After centrifugation, 85-µl aliquots of supernates were injected onto tandem Supelosil C18 columns (Supelco, Bellefonte, PA) heated to 40°C. During elution at a flow rate of 0.5 ml/min, ultraviolet absorbance was monitored at 276 nm. RAPA concentrations were estimated on the basis of a calibration curve consisting of 8 drug-free whole-blood (or tissue) samples that had been spiked with 0, 2, 5, 10, 20, 30, 40, or 50 ng of RAPA. The assays for tissue concentrations added 0.5 ml of drug-containing (or, for calibrators, exogenously spiked) homogenates to 0.5 ml of sodium carbonate. The intra-/interassay coefficients of variation for blood and tissue RAPA measurements were 6.4% at 4.0 ng/ml and 4.2% at 32 ng/ml, and 7.8% at 4.0 ng/ml and 5.6% at 32 ng/ml (38).
Statistical Analyses
Because all immunosuppressive agents studied to date
(39) have obeyed the
median-effect equation of Chou and Talalay
(40), which relates dose (or
concentration) to biologic effect, this model was chosen to assess the
nephrotoxic interactions between CsA and RAPA. The relationship is described
by the following equation:
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Renal Function Changes
Animals that were treated with either CsA or RAPA monotherapy showed
greater urine output than hosts in the control groups
(Figure 1B; P <
0.002), suggesting the presence of renal injury. Because blood glucose levels
were similar among rats in each group, there was no evidence that
hyperglycemia was producing a diuretic effect. In contrast to the control
group, which showed a mean SCr value of 0.25 ± 0.05 mg/dl, treatment
with ascending 2.5 to 20.0 mg/kg per d doses of CsA produced serial increases
in SCr values (all groups, P < 0.0007;
Figure 1C). In contrast, rats
that were treated with the smaller RAPA doses (0.4, 0.8, or 1.6 mg/kg per d)
showed insignificant changes; only animals that received 3.2 or 6.4 mg/kg per
d showed significantly increased SCr values (P = 0.001). The CsA/RAPA
groups showed higher SCr concentrations than either monotherapy group,
increasing from 0.35 ± 0.05 mg/dl for the 2.5/0.4 mg/kg per d group to
2.35 ± 0.37 mg/dl for the 20.0/6.4 mg/kg per d group. Interestingly,
ascending RAPA doses added to fixed amounts of CsA (5 or 10 mg/kg per d)
produced less adverse effects (P = 0.005) than those observed in
groups with simultaneously increasing doses of both drugs (P =
0.001).
The SCr results were confirmed by the GFR values. In comparison to the normal GFR values (1.98 ± 0.34 ml/min; Figure 1D), animals that were treated with CsA doses of 5.0 mg/kg per d (1.1 ± 0.2 ml/min), 7.5 mg/kg per d (0.92 ± 0.37 ml/min), or 20.0 mg/kg per d (0.75 ± 0.24 ml/min) displayed significantly reduced GFR values. Although RAPA alone caused only modest effects, the GFR values were significantly lower among CsA/RAPA combination groups. Thus, RAPA potentiates the dose-dependent nephrotoxicity of CsA (CsA versus CsA/RAPA, P = 0.025 and RAPA versus CsA/RAPA, P = 0.0045).
Blood and Tissue CsA and RAPA Concentrations
Twenty-four h after the last dose of a 14-d course of therapy, CsA and RAPA
concentrations were measured in whole blood as well as in liver and kidney
tissues. Whole-blood trough concentrations increased proportionate to the CsA
dose. At each dose level, concomitant administration of RAPA produced a
significant pharmacokinetic interaction to increase further the CsA
concentrations by approximately twofold above those found in hosts that were
treated with CsA alone (Figure
2A; all, P < 0.006).
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Similarly, the whole-blood concentrations of RAPA, which displayed dose-dependent increases, were exaggerated when the drug was administered concomitantly with CsA (Figure 2B). However, kidney and liver tissue drug concentrations showed even greater degrees of pharmacokinetic interactions among all combination groups (Figure 2, C through F; all, P < 0.001). These findings document that individual drug doses are a poor index of CsA and RAPA exposure in whole blood as well as in tissues when the drugs are used in combination therapy.
Median Effect Analysis of Renal Function
Figure 3 shows a
concentration-dependent increase in SCr values among hosts that were treated
with CsA/RAPA combinations. Because the effect showed the best correlation
with CsA concentrations in kidney tissue samples, it seems likely that the
reduction in renal function was due to the impact of RAPA to increase CsA
levels. Indeed, the pharmacokinetic interaction produced a 12-fold increase in
renal CsA concentrations, compared with the only 4-fold increase in CsA doses.
Figure 4A shows an increase in
SCr upon addition of a fixed 0.8 mg/kg per d dose of RAPA to ascending doses
of CsA (P = 0.0001). However, when the data were expressed as kidney
tissue CsA concentrations, the increase actually was less than would have been
predicted. Thus, RAPA seemed to exert a protective effect
(Figure 4B; P =
0.002).
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A median effect analysis was performed to examine rigorously dose- and concentration-dependent effects of CsA and/or RAPA on renal function, expressed as SCr levels or GFR (Table 1). The Pearson's correlation coefficient values documented a good relation between drug doses and the median effect model for both SCr and GFR values (CsA r = 0.92/RAPA r = 0.76 and CsA r = 0.90/RAPA r = 0.97, respectively). The median effect analysis also showed good correlations between kidney tissue concentrations and SCr levels or GFR (CsA r = 0.90/RAPA r = 0.77 or CsA r = 0.85/RAPA r = 0.98, respectively). Although the CI values that were calculated on the basis of drug doses suggested a synergistic interaction between CsA and RAPA, those that were based on whole-blood concentrations, which were markedly increased by the drug combination, showed less synergism and those that were based on kidney tissue concentrations demonstrated pharmacodynamic antagonism. These findings suggest that the alteration in renal function associated with CsA/RAPA dual-drug therapy primarily represents a pharmacokinetic interaction.
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Renal Analytes
Serum magnesium, uric acid, and phosphate concentrations were measured as
potential indicators of altered tubular function. In a dose-dependent manner,
CsA but not RAPA reduced serum magnesium concentrations (P <
0.00001; data not shown). Interestingly, virtually all animals in the CsA/RAPA
combination groups displayed magnesium concentrations similar to those of
hosts that were treated with RAPA alone. Uric acid concentrations were
increased by CsA monotherapy, especially at 15 and 20 mg/kg per d doses,
although not significantly by RAPA alone. However, all animals in the CsA/RAPA
combination groups displayed a dose-dependent increase in serum uric acid
concentrations even greater than that for animals that were given CsA alone
(P = 0.02 to 0.002; data not shown). A similar picture was observed
for phosphate levels: only hosts that were treated with high doses of CsA
alone displayed increased phosphate levels, whereas animals that received
combination therapy displayed a dose-dependent increase in this analyte. When
corrected for renal tissue concentrations, the effect of the drug combinations
was found to be predominantly additive; only serum phosphate concentrations
showed synergistic interactions (Table
2). Combination therapy had no significant impact on serum sodium
(Table 2) or serum potassium
concentrations (Figure 5). Furthermore, the dose-dependent increase in blood glucose levels produced by
CsA but not by RAPA monotherapy was only slightly greater among the
combination treatment groups. Overall, pharmacokinetic interactions of RAPA to
increase renal tissue CsA concentrations readily accounted for the observed
changes in renal analytes.
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Renal Histology
Kidneys from rats that were given a low-salt diet alone or treated with the
smallest drug doses showed scant evidence of pathologic abnormalities
(Figure 6). Ascending doses
from 0.8 to 6.4 mg/kg per d RAPA alone or 5.0 to 20.0 mg/kg per d CsA alone
were associated with progressive tubular and glomerular abnormalities, as well
as arterial wall thickening. Renal sections from hosts that were treated with
the 10 mg/kg per d CsA dose showed increased glomerular cellularity
accompanied by modest (<25%) thickening of vessels, focal tubular dilation,
and significantly increased interstitial fibrosis. The next higher CsA dose
(15 mg/kg per d) caused significant arteriolar thickening, as well as focal
necrotic changes, tubular dilation, and mild inflammatory cell infiltrates.
The highest CsA dose (20 mg/kg per d) was associated with arteriolar
vacuolization and hyalinosis, glomerular hypercellularity, tubular dilation,
and diffuse interstitial inflammation and fibrosis. In contrast, only moderate
changes in renal histopathology were present even at the highest RAPA dose
(6.4 mg/kg per d).
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Although kidneys from rats that were treated with the combination of 2.5 mg/kg per d CsA and 0.4 mg/kg per d RAPA did not show tubular or glomerular changes, they did display focal vasculitis, including cellular infiltration into the walls of medium- to large-sized vessels. Increasing doses of the CsA/ RAPA combination (5.0/0.8, 7.5/1.2, 10/1.6, 15/3.2, and 20/6.4 mg/kg per d) were associated with progressive tubular damage and thickening of the walls of small arterioles, as well as perivascular infiltration—changes that were far more pronounced than those observed among hosts that were treated with either drug alone. Similar changes were observed among rats that were treated with escalating doses of RAPA (0.4 to 6.4 mg/kg per d) in combination with fixed 5.0 or 10.0 mg/kg per d CsA (not shown). Thus, the addition of RAPA to a CsA regimen accelerates the appearance of histopathologic findings, confirming and extending the observations on alterations of GFR and SCr values.
Effects of CsA and RAPA alone or in Combination on Bone Marrow
Hematopoiesis
Bone marrow cellularity was estimated as the ratio of space occupied by
cellular versus adipose elements
(Figure 7A). In comparison with
the 97.66 ± 2.25% value for cellularity in untreated rats on a low-salt
diet, even the highest doses of CsA (10 to 20 mg/kg per d) displayed little
change (95.33 ± 2.94% to 87.5 ± 4.18%). RAPA alone (0.8 to 6.4
mg/kg per d) caused only a slight dose-dependent decrease in cellularity
(92.83 ± 2.63% to 85.0 ± 6.32%; all, P < 0.001). In
contrast, hosts that were treated with CsA/RAPA combinations displayed more
profound reductions in cellularity, namely to 67.16 ± 26.76% at
10.0/1.6 mg/kg per d and to 54.16 ± 30.72% at 20.0/6.4 mg/kg per d
(CsA/RAPA versus CsA [P < 0.004] versus RAPA
[P < 0.005]). A similar decrease in bone marrow cellularity was
observed when fixed amounts of CsA (5 or 10 mg/kg per d) were combined with
ascending RAPA doses (all, P < 0.001). These interactions were
modestly synergistic, whether the analysis was performed on the basis of drug
doses or of renal (or liver) concentrations as a surrogate for bone marrow
levels (Table 3).
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Control rats on a low-salt diet displayed 14.8 ± 3.3 megakaryocytes (MEG) per high-power (40x) field (HPF). Monotherapy with 10.0 mg/kg per d CsA significantly decreased the values to 9.75 ± 1.64 MEG/HPF, 15.0 mg/kg per d CsA to 9.66 ± 3.38 MEG/HPF, 3.2 mg/kg per d RAPA to 10.83 ± 1.60 MEG/HPF, and 6.4 mg/kg per d RAPA to 9.33 ± 1.75 MEG/HPF (all, P < 0.0001; Figure 7B). A greater effect was produced by CsA/RAPA combinations: 7.5/1.2 mg/kg per d to 9.83 ± 0.75 MEG/HPF and 15.0/3.2 mg/kg per d to 7.0 ± 0.89 MEG/HPF. The median effect analysis showed a range of interactions between CsA and RAPA spanning from synergistic to additive in nature, whether expressed as dose, whole-blood, or kidney tissue concentrations (Table 3). In contrast, the decrease in peripheral blood leukocyte counts associated with RAPA consistently showed an antagonistic interaction of concomitant CsA therapy. Overall, these findings suggest that addition of CsA significantly and probably synergistically exacerbates RAPA-induced myelosuppression.
Effect of CsA and RAPA alone or in Combination on
Cholesterol-Containing Lipoprotein Fractions
RAPA monotherapy significantly increased the content of both low-density
lipoprotein (LDL) cholesterol and serum cholesterol values in comparison with
the modest increase induced by CsA alone. CsA/RAPA combination groups showed a
significant interaction to increase LDL cholesterol
(Figure 8A) but only modest
effects on serum cholesterol (Figure
8B). These trends were confirmed by the median effect analysis
(Table 2). In contrast, RAPA
monotherapy caused a significant dose-dependent increase in high-density
lipoprotein cholesterol levels, whereas both CsA monotherapy and CsA-RAPA
combination therapy produced only modest effects (P = 0.0002;
Figure 8C). Thus, the property
of RAPA that causes increased lipoprotein cholesterol levels seems to be
augmented by a dynamic interaction with CsA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We sought to compare the effects of ascending doses of each drug alone versus in combination in salt-depleted rats, an animal model that is not complicated by the occurrence of allograft rejection. We confirmed that CsA administration in this model caused functional and histopathologic renal changes similar to those observed in patients who undergo chronic drug administration (1,43). We also observed that salt-depleted rats that were treated with RAPA alone displayed reduced GFR values only at high doses, a finding that was consistent with the previous work of Andoh et al. (22) and of DiJoseph et al. (24). Although the present findings of exaggerated reduction in renal function among salt-depleted rats that were treated with combinations of RAPA and CsA concur with the observations of Andoh et al. (22), Andoh et al. ascribed their findings to hyperglycemia, whereas we documented that it was due to potent pharmacokinetic interactions.
We demonstrated elsewhere that pharmacokinetic interactions potentiate the synergistic immunosuppressive effects of CsA and RAPA in rats (26) and that similar effects dramatically increase the whole-blood and, especially, the renal tissue concentrations of each agent, confirming and extending observations of Napoli et al. (27) in normal rats and those of Kaplan et al. (44) in renal transplant patients. The drug interaction may owe to mutual competitive interactions of both drugs as substrates for P-glycoprotein and for cytochrome P450 isozymes (45). The increases in concentration correlated with the observed alterations in SCr and GFR. Indeed, analysis of the adverse effects on the basis of concentrations rather than doses of CsA showed that the addition of RAPA actually mitigated the nephrotoxic injury, compared with that anticipated on the basis of the observed drug exposure. These findings suggest that the development of concentration-control algorithms may enable clinicians to minimize the renal injury associated with CsA/RAPA regimens. Indeed, it seems likely that the blinded design of the pivotal trials, which demanded the use of therapeutic CsA exposures in both the Aza and the placebo groups to protect the graft, may have led inadvertently to toxic CsA levels in the kidney transplants of the RAPA cohorts. Indeed, a logical extension of the present findings suggests that it may be more appropriate to combine doses of the non-nephrotoxic agent RAPA that are relatively higher than were administered in the pivotal trials with reduced doses of the nephrotoxic agent CsA, to exploit the synergistic immunosuppressive interactions and thereby minimize potentially toxic pharmacokinetic interactions.
Because RAPA inhibits transduction cascades evoked by a variety of cytokine signals, it may antagonize the nephrotoxicity of CNA by blocking the effects of a local humoral mediator such as angiotensin II (46). On the one hand, the production of both angiotensin II itself and its receptor is upregulated by CsA. On the other hand, angiotensin II receptor-mediated signal transduction depends on p70s6 kinase, a product downstream from the action of mammalian target of rapamycin. Future studies to evaluate the content and activity of this cytokine and other humoral mediators will be critical to dissect the molecular basis of drug interactions in the kidney.
In contrast to the predominant role of pharmacokinetic interactions to produce renal dysfunction in animals that were treated with CsA/RAPA, there seemed to be more than additive effects of CsA to exacerbate the myelosuppressive and hypercholesterolemic toxicities produced by RAPA. The significant dose- and concentration-dependent myelosuppressive effects of RAPA were not shared by CsA. This toxicity presumably is related to the inhibitory effect of RAPA on the mammalian target of rapamycin, which catalyzes an essential step in the signal transduction pathway of proliferation-responsive cell lines upon addition of interleukin-3 (IL-3), IL-6, granulocyte colony stimulating factor (CSF), and granulocyte-macrophage CSF—all critical factors for hematopoiesis. The complementary actions of CsA to reduce production of at least some of these cytokines, including IL-3 and granulocyte-macrophage CSF, may potentiate the transduction blockade produced by RAPA. Although doses as high as 50 mg/kg per d RAPA had no effect on myelopoiesis in normal mice, Quesniaux et al. (47) observed impaired hemopoietic recovery during the 10-d period after injection of 5-fluorouracil. In humans, RAPA reduces the content of newly formed blood elements in a dose- and concentration-dependent manner, particularly during the early posttransplantation period, possibly because of the higher drug doses used during the induction phase and other associated perioperative stressors (48).
A similar pharmacodynamic interaction with CsA seemed to exacerbate hyperlipidemia, a well-recognized side effect of RAPA (49,50). In contrast to CsA, which seems to block LDL hepatic clearance mechanisms, RAPA displays a distinctive effect to reduce lipoprotein lipase activity in humans (Morrisett JD, Kahan BD, unpublished observations). Because the mammalian target of rapamycin is an intermediate in the pathway of insulin-driven release of lipoprotein lipase activity (Knutsen P, Kahan BD, unpublished observations), the increased LDL cholesterol may owe, at least in part, to dual sites of action of the two drugs.
On the basis of the present study, which shows that the effect of RAPA to potentiate drug-induced nephrotoxicity owes to increased CsA concentrations in both blood and kidney tissue—a pharmacokinetic interaction—clinicians must recognize that whole-blood levels do not reflect renal tissue levels. Indeed, these findings suggest that increasing RAPA exposure may permit substantial CsA reduction, thereby mitigating drug-induced nephrotoxic effects. In contrast, the effects of CsA to exacerbate RAPA-induced toxicities of myelosuppression and of hypercholesterolemia seem to be concentration-independent pharmacodynamic interactions, which were greater than those anticipated from the observed changes in whole-blood or tissue drug levels. Recent clinical experience suggests that drug doses should be adjusted across a therapeutic range of RAPA (and CsA) concentrations according to the patient's proclivity to adverse effects: early posttransplantation in the presence of impaired renal function, we now seek to achieve trough concentration ranges of RAPA = 10 to 20 ng/ml and CsA = 50 to 100 ng/ml. Alternatively, in the presence of myelosuppression or hyperlipidemia, we individualize drug doses to obtain concentration ranges of 5 to 15 ng/ml RAPA and 125 to 175 ng/ml CsA. Clearly, incisive molecular analyses of critical cytokine content/effects in experimental animals are urgently required to guide the formulation of insightful concentration-controlled, randomized clinical trials.
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); RAPA alone at
doses of 0.4, 0.8, 1.2, 1.6, 3.2, or 6.4 mg/kg per d (
); or CsA/RAPA
combination at doses of 2.5/0.4, 5.0/0.8, 7.5/1.2, 10.0/1.6, 15.0/3.2, or
20.0/6.4 mg/kg per d ([UNK]). In addition, some rats were treated with a
constant dose of 5 ([UNK]) or 10 ([UNK]) mg/kg per d CsA with ascending RAPA
doses, namely 0.4, 0.8, 1.2, 1.6, 3.2, and 6.4 mg/kg per d. After 14 d, we
measured percentage of weight change in comparison with the weight at the
beginning of therapy (A), 24-h urine output (B), serum creatinine levels (SCr;
C), and GFR (D). For details, see Materials and Methods section.
