Identification of gallic acid as a active ingredient of Syzygium aromaticum against tacrolimus-induced damage in renal epithelial LLC-PK1 cells and rat kidney.

Tacrolimus (FK506), a calcineurin inhibitor, is an effective immunosuppressive agent mainly used to lower the risk of organ rejection after allogeneic organ transplant. However, FK506-associated adverse effects, such as nephrotoxicity, may limit its therapeutic use. In this study, we confirmed that epigallocatechin-3-gallate (EGCG), sanguiin H-6, and gallic acid increased cell survival following FK506-induced cytotoxicity in renal epithelial LLC-PK1. Among these compounds, gallic acid exerted the strongest protective effect, further confirmed in the FK506-induced nephrotoxicity rat model. Additionally, we identified supporting evidence for the nephroprotective function of gallic acid using molecular docking and bioavailability investigations.

Ginseng extract reduces tacrolimus-induced oxidative stress by modulating autophagy in pancreatic beta cells.

We previously reported that long-term treatment with a calcineurin inhibitor impairs autophagy process in pancreatic beta cells. This study investigated the effect of Korean red ginseng extract (KRGE) on autophagy modulated by oxidative stress. In mice with tacrolimus (Tac)-induced diabetes mellitus, KRGE alleviated islet dysfunction and decreased oxidative stress and autophagic vacuoles. In vitro, KRGE decreased autophagosome formation and attenuated lysosomal degradation, accompanied by improved beta cell viability and insulin secretion. Addition of 3-methyladenine (3-MA), an inhibitor of autophagosomes, to KRGE further improved cell viability and insulin secretion, and bafilomycin A (BA), an inhibitor of lysosomal function, reduced the effects of KRGE. At the subcellular level, Tac caused mitochondrial dysfunction (impaired mitochondrial oxygen consumption, ATP production, and increased reactive oxygen species production). But KRGE improved these parameters. The effect of KRGE on mitochondrial function enhanced by 3-MA but decreased by BA, suggesting a causal relationship between KRGE effect and autophagy modulation in Tac-induced mitochondrial dysfunction. These findings indicate that KRGE modulates autophagy favorably by reducing Tac-induced oxidative stress, and this effect is closely associated with improvement of mitochondrial function.

Tacrolimus Triggers Transient Receptor Potential Vanilloid-1-Dependent Relapse of Pancreatitis-Related Pain in Mice.

Transient receptor potential vanilloid-1 (TRPV1) expressed in nociceptors is directly phosphorylated and activated by protein kinase C, and involved in the signaling of pancreatic pain. On the other hand, Cav3.2 T-type Ca2+ channels expressed in nociceptors are functionally upregulated by phosphorylation with protein kinase A and also play a role in pancreatitis-related pain. Calcineurin, a phosphatase, negatively regulates various channel functions including TRPV1, and calcineurin inhibitor-induced pain syndrome by tacrolimus, a calcineurin inhibitor, used as an immunosuppressant, has been a clinical problem. We thus examined the effect of tacrolimus on pancreatitis-related pain in mice. Repeated treatment with cerulein caused referred hyperalgesia accompanying acute pancreatitis, which was unaffected by tacrolimus. Pancreatitis-related symptoms disappeared in 24 h, whereas the referred hyperalgesia recurred following the administration of tacrolimus, which was abolished by the blockers of TRPV1 but not T-type Ca2+ channels. Thus, tacrolimus appears to cause the TRPV1-dependent relapse of pancreatitis-related pain, suggesting the involvement of calcineurin in the termination of pancreatic pain.

The Protective Effect of Febuxostat on Chronic Tacrolimus-Induced Nephrotoxicity in Rats.

BACKGROUND: The use of calcineurin inhibitors is a well-known risk factor for hyperuricemia in kidney transplant recipients. We evaluated the effect of febuxostat (Fx), a new uric acid-lowering drug, on hyperuricemia and renal injury in an experimental model of chronic tacrolimus (Tac)-induced nephropathy. METHODS: Chronic Tac nephropathy was induced by administering Tac (1.5 mg/kg/day) to rats on a low-salt diet (0.05%) with oxonic acid (OA, 2%, 0.2 g/kg/day) for 28 days. Two doses of Fx (5 and 10 mg/kg) were concomitantly administered with Tac or vehicle (Vh). We evaluated the effect of Fx on hyperuricemia by measuring serum uric acid (SUA) levels, fractional excretion of uric acid (FEUA), and urate transporters in Tac-induced nephropathy. The effects of Fx on Tac-induced renal injury were evaluated in terms of renal function and arteriolopathy, tubulointerstitial fibrosis, inflammation, and apoptosis. We evaluated oxidative stress as a protective mechanism via xanthine oxidase (XO) activity, and as a marker of oxidative stress (via evaluation of levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxy-2-hexenal (4-HHE)). RESULTS: The Tac group showed higher SUA levels and lower FEUA than did the Vh group, but Fx treatment significantly decreased SUA levels in a dose-dependent manner, with an increase of FEUA at a high dose (10 mg/kg). Tac treatment increased urate-anion exchanger 1 and decreased organic anion transporter type 1 expression in renal tubular cells, but Fx treatment reversed the effects on those transporters. Impaired renal function and histological injury (interstitial fibrosis, inflammation, and arteriolopathy) in the Tac group were markedly improved by Fx administration. Increases in apoptotic cell death and activation of proapoptotic caspase-3 by Tac were remarkably decreased by Fx treatment. Tac administration increased the activity of XO in kidney tissue and serum, and the levels of 8-OHdG in urine and 8-OHdG and 4-HHE of kidney tissue, but combined treatment with Fx decreased the levels of these parameters. CONCLUSIONS: Fx is effective in controlling hyperuricemia and in preventing Tac-induced renal injury, via a reduction of oxidative stress. Therefore, a targeted therapy aimed at inhibiting uric acid by Fx may be a useful approach in the management of the progression of nephropathy in renal transplant patients treated with Tac.

Protective effect of mycophenolate mofetil against nephrotoxicity and hepatotoxicity induced by tacrolimus in Wistar rats.

Tacrolimus (TAC), a calcineurin inhibitor (CNI), is clinically used as an immunosuppressive agent in the transplant recipient; however, the use of TAC is greatly limited by its nephrotoxicity and hepatotoxicity. Mycophenolate mofetil (MMF), an inhibitor of the purine synthesis, has been used in combination with many immunosuppressive drugs such as TAC. The association TAC/MMF was used in organ transplantation to increase the efficiency and reduce acute rejection rates, but the effects of MMF on TAC-induced kidney and liver injuries are still not well investigated. The aims of this study are to explore whether MMF co-administration with TAC has a renoprotective and hepatoprotective effect against TAC-induced renal and hepatic injuries and to check the implication of oxidative stress in the MMF's possible protective effect. Our results showed that MMF (at 50 mg kg(-1) body weight (b.w.)) restored creatinine, in addition to increased AST and ALT levels by TAC (at 60 mg kg(-1) b.w.). Furthermore, MMF decreased DNA damage induced by TAC in the kidney and liver of rats as assessed by comet assay. This renoprotective and hepatoprotective effect of MMF was associated with an antioxidant effect. In fact, MMF co-treatment with TAC decreased oxidative damage induced by TAC. It reduced malondialdehyde (MDA) and protein carbonyl (PC) levels as well as catalase and superoxide dismutase (SOD) activities. We conclude that the co-administration MMF with TAC protect liver and kidney against TAC toxicity via an antioxidant process.

Inhibition of dipeptidyl peptidase IV protects tacrolimus-induced kidney injury.

Accumulating evidence shows that a gut-released hormone, the glucagon-like peptide-1 (GLP-1), has not only a glucose-lowering effect but also a renoprotective effect against kidney injury. In this study, we investigated whether a dipeptidyl peptidase (DPP) IV inhibitor has a protective effect against tacrolimus-induced renal injury. Rats were treated with tacrolimus (1.5 mg/kg, subcutaneously) and the DPP IV inhibitor MK0626 (10 or 20 mg/kg, oral gavage) for 4 weeks. MK0626 treatment attenuated tacrolimus-induced renal dysfunction, tubulointerstitial fibrosis, and arteriolopathy. Moreover, these improvements were accompanied by a reduction in oxidative stress and apoptosis. MK0626 treatment increased the blood level of GLP-1 and the level of its receptor in tissue sections but did not alter the levels of other DPP IV substrates, such as neuropeptide Y and the stromal cell-derived factor-1. These data suggest that DPP IV inhibition has an important role in the renoprotection against tacrolimus-induced nephrotoxicity via antioxidative and antiapoptotic effects and preservation of the GLP-1 system.

Tacrolimus-induced hypertension and nephrotoxicity in Fawn-Hooded rats are attenuated by dual inhibition of renin-angiotensin system.

Chronic immunosuppressive therapy is often complicated by the development of both arterial hypertension and renal dysfunction. The principal aim of this study was to assess the effects of dual inhibition of renin-angiotensin system (RAS) and other antihypertensive treatment on blood pressure and renal function in normotensive and hypertensive Fawn-Hooded (FH) strains during chronic calcineurin inhibitor (CNI) administration. Combinations of perindopril (5 mg kg(-1) per day) and losartan (50 mg kg(-1) per day) or amlodipine (6 mg kg(-1) per day) and metoprolol (80 mg kg(-1) per day) were administered to normotensive (FHL) and hypertensive (FHH) rats, fed with diet containing tacrolimus (Tac; 12 mg kg(-1) per day). Tac-induced arterial hypertension in both animal strains (FHL: 151±4; FHH: 198±6 mm Hg) was prevented by dual RAS inhibition (FHL: 132±3 mm Hg, P<0.05; FHH: 153±3 mm Hg, P<0.05) as well as by a combination of amlodipine and metoprolol (FHL: 136±3 mm Hg, P<0.05; FHH: 166±4 mm Hg, P<0.05). However, significant nephroprotection was observed only in animals on dual RAS inhibition where albuminuria was reduced in both FHL (51.1±3.9 vs. 68.3±4.5 µg per day; P<0.05) and FHH rats (13.1±0.3 vs. 18.8±0.7 mg per day; P<0.05). We also found Tac-induced enhancement in renal angiotensin II activity that was significantly reduced by dual RAS inhibition in both FHL (63.5±3.2 vs. 23.1±3.0 fmol g(-1)) and FHH (79.8±8.5 vs. 32.2±5.8 fmol g(-1)). In addition, histological analysis revealed that RAS inhibition noticeably diminished glomerulosclerosis and tubulo-interstitial injury. This study indicates that dual blockade of RAS significantly attenuates Tac-induced arterial hypertension and nephrotoxicity in FH rats and further supports the notion that RAS inhibitors display efficient renoprotective properties during CNI treatment.

Costimulatory blockade with mTor inhibition abrogates effector T-cell responses allowing regulatory T-cell survival in renal transplantation.

The advent of novel immunosuppressive strategies in renal transplantation, with immunomodulatory properties, might facilitate long-term allograft survival. T-cell depletion, costimulation-blockade and mTor inhibition have been shown to favour anti-donor hyporesponsiveness. Recently, the combination of rATG, belatacept (Bela) and sirolimus (SRL) has been used in kidney transplantation, showing very low incidence of acute rejection and excellent 12-month graft and patient survival. Herein, we have analysed the 1-year evolution of memory/effector and regulatory T cells and assessed the donor-specific T-cell alloimmune response in a group of these patients and compared with others treated with a calcineurin-inhibitor(CNI)-based (rATG/tacrolimus/MMF), and two other Bela-based regimens (rATG/Bela/MMF and basiliximab/Bela/MMF/steroids). During the first year after transplantation, patients receiving rATG/Bela/SRL had significantly higher percentage of Tregs upon the memory T-cell compartment and showed a potent anti-donor suppressive activity. In an in vitro naive and memory/effector T-cell co-culture, the combination of costimulation-blockade and SRL could abrogate both antigen-specific T-cell responses as efficiently as using a CNI drug. The combination of T-cell depletion, costimulation-blockade and mTor inhibition seems to be able to allow Treg survival and inhibit donor-specific alloreactive effector immune responses after kidney transplantation in humans.

Ultrastructural alterations in endothelial mitochondria are associated with enhanced nitrotyrosine accumulation and progressive reduction of VEGF expression in sequential protocol renal allograft biopsies with calcineurin inhibitor toxicity.

Calcineurin inhibitors (cyclosporine and tacrolimus; CNIs) continue to be used as constituents of post-transplant immunosuppression in most centers. However, renal toxicity associated with the use of these drugs remains a problem adversely affecting the long-term graft survival. Fifteen adequate protocol renal allograft biopsies, with histological features of CNI toxicity among 140 protocol biopsies performed at 1-, 6-, and 12-month post-transplant, were included. Mitochondrial alterations in the tubular epithelial cells and endothelia of glomerular, peritubular capillaries and arterioles were graded semiquantitatively and further ultrastructural morphometric evaluation of numerical density and area of the mitochondria was performed. Immunohistochemical staining for nitrotyrosine (marker of peroxynitrite formation) and vascular endothelial growth factor (VEGF) was performed and expression graded semiquantitatively. Higher grades of alterations were seen in endothelial mitochondria as compared with tubular mitochondria in biopsies with calcineurin inhibitor toxicity (CNIT). Endothelial mitochondrial numerical density showed progressive decline over 1-, 6- and 12-month biopsies while area showed progressive increase in biopsies with CNIT as compared with controls. Upregulation of nitrotyrosine was seen even at 1-month post-transplant, persisted at 6 and 12 months, and was significantly greater than that in control biopsies. Intense VEGF expression was noted in early CNIT while progressive reduction was seen in 6- and 12-month protocol biopsies. This study shows a relatively high incidence of CNIT in protocol renal allograft biopsies, indicating that this might be an important mechanism of background damage to the allograft. Structural alterations in endothelial mitochondria are consistent findings in protocol biopsies with CNIT and this relatively specific mitochondrial damage may stem from the peroxynitrite-mediated damage associated with progressive loss of protective function of VEGF.

Voriconazole inhibition of tacrolimus metabolism in a kidney transplant recipient with fluconazole-resistant cryptococcal meningitis.

Fluconazole resistance among Cryptococcus neoformans is unusual in post-transplantation patients. Voriconazole is a triazole agent with good antifungal activity but also with drug-drug interactions because of potent inhibition of the P450 enzyme system. The interaction with immunosuppressive agents, especially calcineurin inhibitors, is of concern in post-transplantation patients. We report the first case of fluconazole-resistant cryptococcal meningitis in a kidney transplant recipient successfully treated with voriconazole, but complicated with a raised serum concentration of tacrolimus and hyponatremia after co-administration. A 43-year-old man with a history of renal transplantation and on long-term immunosuppressive agents, including mycophenolate and tacrolimus, suffered from recurrent cryptococcal meningitis. He was treated with amphotericin B-liposome for 24 days because of fluconazole resistance. However, cryptococci were still found in the cerebrospinal fluid; oral voriconazole was substituted. Six days after co-administration of voriconazole and tacrolimus, the trough concentration of tacrolimus markedly increased and hyponatremia developed. A culture of the CSF did not yield growth of Cryptococcus. Conditions improved after the cessation of tacrolimus for three days followed by reducing the dosage of voriconazole and tacrolimus. When voriconazole is initially added, the dosage of tacrolimus should be reduced. Close monitoring of tacrolimus concentration and its adverse effects, including nephrotoxicity, hyperglycemia, hyperkalemia, and hyponatremia, are mandatory.

Drug-drug interaction in a kidney transplant recipient receiving HIV salvage therapy and tacrolimus.

Concomitant use of immunosuppressive agents and antiretroviral drugs may lead to complex drug-drug interactions. The calcineurin inhibitor tacrolimus is metabolized by cytochrome P-450 3A4 (encoded by the CYP3A4 gene) and is a substrate of P-glycoprotein (encoded by the ABCB1 gene). Both pathways can be inhibited by protease inhibitors (PIs). The reduction in first-pass and postabsorptive metabolism of tacrolimus by PIs can lead to extreme prolongation of the elimination half-life and significantly increase tacrolimus trough levels. In a patient with human immunodeficiency virus (HIV)-associated focal segmental glomerulosclerosis leading to kidney cadaveric transplantation, HIV salvage therapy was started with the new PI darunavir and boosted with ritonavir, another PI. The reduction in first-pass and postabsorptive metabolism of tacrolimus by PIs led to a dramatic increase in tacrolimus trough levels and extreme prolongation of the elimination half-life. Trough levels of tacrolimus levels were as high as 106.7 ng/mL. A decrease in tacrolimus dosage to a single dose of 0.5 mg/wk, corresponding to 3.5% of the usual dose, enabled maintenance of stable tacrolimus trough levels. Our case highlights that coadministration of a PI and tacrolimus is feasible through intense reduction in dose and prolongation of the dosing interval of the calcineurin inhibitor. Complex drug interactions may become more frequent because more HIV-infected patients are undergoing transplantation and newer HIV drugs are being used. Close monitoring and excellent adherence are mandatory to avoid the risk of harm for the graft and patient.

Everolimus and sirolimus antagonize tacrolimus based calcineurin inhibition via competition for FK-binding protein 12.

The calcineurin inhibitors cyclosporin A and tacrolimus and the inhibitors of the mTOR, sirolimus and everolimus bind immunophilins that are required for their immunosuppressive action. In contrast to cyclosporin A, tacrolimus and the mTOR inhibitors (MTIs) share common immunophilins, the FK506-binding proteins (FKBPs). We investigated the immunosuppressive interactions of MTIs on tacrolimus based immune suppression, since insights in immunological drug-drug interactions can be very relevant for optimization of immunosuppressive regimens in allograft transplantation medicine. Isolated peripheral blood mononuclear cells from healthy volunteers were incubated with combinations of MTIs and calcineurin inhibitors and when monitored for calcineurin activity and IL-2 excretion after mitogen stimulation, tacrolimus IC(50) concentrations shifted to higher concentrations in the presence of MTIs. This antagonism was absent for cyclosporin A, reproducible for 10 healthy volunteers (p<0.001) and stronger for sirolimus than for everolimus. When cell lysate was treated with and without MTI, tacrolimus and FKBP12, FKBP12 could increase calcineurin inhibition by tacrolimus and reverse the MTI antagonism for both MTIs. These results demonstrate that FKBP12 can be rate limiting for calcineurin inhibition at high tacrolimus concentrations and that the antagonism of sirolimus and everolimus on tacrolimus based immune suppression is mediated via saturation of FKBP12.

Effects of tacrolimus (FK506) on the development of osteonecrosis in a rabbit model.

The present study examined the effects of tacrolimus (FK506) on the development of osteonecrosis in rabbits. In Experiment A, rabbits were given FK506, and also given a single dose of steroid. Control rabbits were given the same dose of steroid only. In Experiment B, rabbits were given FK506 and a reduced dose of steroid. The results showed that addition of FK506 did not change the number of rabbits with osteonecrosis when an identical steroid dose was given. When the steroid dose was reduced, the osteonecrosis incidence significantly decreased (p < 0.01). These results suggest that the clinically reported decrease in the osteonecrosis incidence following the introduction of FK506 is most likely attributable to the lower doses of steroids.

Possible therapeutic effect of lipid supplementation on neurological complications in liver transplant recipients.

Neurological complications (NCs) represent a serious problem following liver transplantation and may develop either because of various peri-operative factors or the toxicity of immunosuppression. Although the causality assessment of NCs can be particularly difficult in the setting of organ transplantation, calcineurin inhibitors (CNIs) might influence NCs to a certain extent, regardless of the etiology. Therefore, minimizing the influence of CNIs could be a reasonable strategy for alleviating NCs. Based on our hypothesis that lipid supplementation prevents lipophilic CNIs from crossing the blood-brain barrier, soybean oil was administered to five liver transplant patients with NCs. In all of these patients, the neurological symptoms improved without discontinuing or reducing the dose of CNIs. Thus, lipid supplementation might be able to reduce the adverse neurological effects of CNIs.

Raloxifene does not affect the changes in bone histomorphometric parameters induced by low dose tacrolimus in male rats.

Raloxifene is a selective estrogen receptor modulator. The drug reduces bone loss and prevents fractures in postmenopausal women. Tacrolimus, an immunosuppressant, is used to prevent organ transplant rejection. The effect of raloxifene and tacrolimus on the osseous bone in men has not been exhaustively determined. To study the effects of raloxifene, tacrolimus as well as concurrent administration of raloxifene and tacrolimus on the osseous tissue in male rats, a preliminary assessment of the drug action on histomorphometric parameters of rat bones was made. The experiments were carried out on mature male Wistar rats. The animals were divided into six groups, 7 animals each: I--control rats; II--rats which were administered raloxifene (5 mg/kg po daily); III-- rats which were administered tacrolimus (0.3 mk/kg po daily); IV - rats which were administered tacrolimus (0.6 mg/kg po daily; V-- rats which were administered raloxifene (5 mg/kg po daily) and tacrolimus (0.3 mg/kg po daily); VI - rats which were administered raloxifene (5 mg/kg po daily) and tacrolimus (0.6 mg/kg po daily). The drugs were administered for 4 weeks. Body mass, macrometric parameters of the tibia, femur and L-4 vertebra, histomorphometric parameters of tibia (transverse growth, width of osteoid, area of the transverse cross section of bone marrow cavity and cortical bone), and the femur (width of trabeculae, width of epiphyseal cartilage) were examined. The action of raloxifene in male rats was demonstrated through increased width of osteoid. An increased traverse growth of bone and osteoid width as well as transverse cross section of the cortical bone and the marrow cavity and increased thickness of trabeculae were observed in male rats receiving tacrolimus at 0.3 mg/kg. The administration of tacrolimus at 0.6 mg/kg resulted in increased traverse growth of bone and increased thickness of osteoid, whereas the thickness of trabeculae remained unaffected. The results obtained in the rats administered concurrently raloxifene and tacrolimus (at 0.3 mg/kg or at 0.6 mg/kg) were similar to those obtained in the group of rats receiving tacrolimus at 0.3 mg/kg. It seems that the most valuable in entire experimental system of the study are the results obtained in the group receiving tacrolimus at 0.3 mg/kg po, which are indicative of intensified bone remodeling processes with dominant the bone formation process.

Protective effect of calcineurin inhibitors on acoustic injury of the cochlea.

This study examined the effect of immunosuppressants, cyclosporin A, FK506 and rapamycin on functional recovery of the cochlea after acoustic overexposure, in guinea pigs and mice. Thirty guinea pigs were exposed to a 2 kHz pure tone at 120 dB SPL for 10 min. The compound action potential threshold shift induced by acoustic overexposure was examined. Twenty-five mice were exposed to a 4 kHz pure tone at 128 dB SPL for 4h. Auditory brainstem response was used to examine the hearing threshold shift. In both the guinea pig and mouse experiments, cyclosporin A and FK506, intraperitonally given just before acoustic overexposure, significantly decreased the hearing threshold shift one or two weeks after acoustic overexposure. However, neither rapamycin nor the FK506 and rapamycin combined treatment groups showed improvement of the threshold shift. The present findings suggest that these two calcineurin inhibitors have a protective effect against acoustic injury of the cochlea, whereas the non-calcineurin inhibitor, rapamycin, not only has no effect against acoustic injury, but rather blocked the effect of FK506. This indicated a possible role of calcineurin against acoustic injury.

Tacrolimus and posttransplant diabetes mellitus in renal transplantation.

The most prominent side effect of tacrolimus is the induction of posttransplant diabetes mellitus (PTDM). In this review, the authors discuss the incidence, mechanism, prevention, and treatment of tacrolimus-induced PTDM in renal patients.

Prograf produces a molecular environment favoring antifibrosis, an effect reversed by the addition of rapamune.

Rapamune, an inhibitor of the mammalian target of rapamycin, exhibits antiproliferative actions and is increasingly used as adjuvant therapy with calcineurin inhibitors. This study investigated the effect of Rapamune on functional and molecular markers in a rat model of calcineurin inhibitor-induced graft dysfunction. Prograf (6 mg), with or without addition of Rapamune (1 mg), was administered to salt-depleted male rats (n = 6/group). Urinary protein excretion and serum creatinine were measured. Rats were culled at 28 days, and messenger RNA expression of TGF-beta, MMP-2, MMP-9, TIMP-1, and collagen III was evaluated with reverse transcriptase polymerase chain reaction. Serum creatinine increased with Prograf (P = .01), but not Rapamune (P = .69) treatment, compared to controls at 28 days. The combination of Rapamune and Prograf produced a rise in serum creatinine at 7 (P = .007) and 14 (P = .01) days, but this was not observed at later time points. Urinary protein excretion was unaltered by any drug or combination. While confirming a synergistic effect of Rapamune and calcineurin inhibitors on renal function, these results suggest that sole therapy with Prograf produces inhibition of fibrotic gene expression. Rapamune alone has no deleterious effect on gene expression but addition of Rapamune cancels out the beneficial effects of Prograf.