New-onset diabetes mellitus (DM) after transplantation (NODAT) is a serious metabolic complication after kidney transplantation, and it is associated with poor patient and graft survival.1 Among several NODAT risk factors, immunosuppressive drugs increase the occurrence risk of NODAT, and tacrolimus (TAC) is an important risk factor of NODAT.2-5 NODAT caused by TAC is partly related to its direct toxic effect on pancreatic islet cells, and oxidative stress plays a major role in TAC-induced pancreas dysfunction.6,7
Cytotoxic T lymphocyte–associated protein 4 immunoglobulin (CTLA4Ig) is a fusion protein that selectively blocks T-cell activation by inhibiting CD28-B7 costimulation.8 Belatacept, a CTLA4Ig, is a promising replacement of calcineurin inhibitors (CNIs), and it helps in limiting CNI-induced nephrotoxicity in kidney transplantation.9 In clinical trials, conversion of from cyclosporine (CsA) or TAC treatment to treatment with belatacept ameliorated allograft dysfunction caused by CNIs.10,11 However, the effect of conversion to CTLA4Ig on TAC-induced DM has not been studied.
Therefore, we examined aspects of conversion to CTLA4Ig treatment on TAC-induced pancreatic islet injury. First, we evaluated the diabetogenicity of CTLA4Ig. Second, we investigated the effect of conversion from TAC to CTLA4Ig treatment on TAC-induced DM in rats. Third, we observed the direct effect of CTLA4Ig on TAC-induced pancreatic islet cell injury. The results of our study may provide a rationale for the use of CTLA4Ig in NODAT caused by TAC.
MATERIALS AND METHODS
Animal Care and Drug Treatment
The Animal Care Committee of the Catholic University of Korea approved the experimental protocol (CUMC-2016-0061-02), and all procedures were performed in strict accordance with the recommendations of the ethical guidelines for animal studies. Male Sprague-Dawley rats (Charles River Technology, Seoul, Korea), initially weighing 220 to 230 g, were housed in cages (Nalge Co., Rochester, NY) in a controlled temperature and 12-hour light/12-hour dark cycle environment. The rats received a 0.05% salt diet (Tekland Premier, Madison, WI). TAC (Prograft, Astellas Pharma Inc., Ibaraki, Japan) was diluted in olive oil (Sigma-Aldrich, St. Louis, MO) to a final concentration of 1.5 mg/kg. CTLA4Ig was generously provided by Bristol-Myers-Squibb (New York, NY) and was dissolved in saline to a final concentration of 0.25, 0.5, 1, 2, and 4 mg/kg.
The first study was designed to evaluate whether CTLA4Ig had a diabetogenic effect in rats. We tested the dose dependency of CTLA4Ig (0.25, 0.5, 1, 2, and 4 mg/kg per week intravenously) for 4 weeks in 9 animals in each group based on survival data in animal studies.12-14 The second study aimed to evaluate the effect of conversion from TAC to CTLA4Ig treatment on pancreatic islet function in TAC-induced DM. After acclimatization and a low-salt diet for 1 week, weight-matched rats were randomized into 2 groups containing 9 rats for the vehicle (VH) group and 36 rats for the TAC group, and were treated daily with VH or TAC for 3 weeks, respectively. After 3 weeks of TAC treatment, we performed intraperitoneal glucose tolerance test (IPGTT) to confirm the development of TAC-induced DM. Thereafter, we divided rats randomly into 3 groups (TAC was continued, withdrawn, or replaced by CTLA4Ig). We injected VH or TAC daily via subcutaneous route, and we injected CTLA4Ig weekly via the tail vein. The study groups were as follows:
- (I) VH group (n = 9): Treatment with olive oil (1 mg/kg per day) for 6 weeks;
- (II) TAC group (n = 9): Treatment with TAC (1.5 mg/kg per day) for 6 weeks;
- (III) TAC withdrawal (TW) group (n = 9): Treatment with TAC (1.5 mg/kg) for 3 weeks, and TAC was withdrawn for the remaining 3 weeks;
- (IV) Conversion from TAC to 1 mg/kg of CTLA4Ig (TC1) group (n = 9): Treatment with TAC (1.5 mg/kg) for the first 3 weeks and CTLA4Ig (1 mg/kg) for additional 3 weeks;
- (V) Conversion from TAC to 2 mg/kg of CTLA4Ig (TC2) group (n = 9): Treatment with TAC (1.5 mg/kg) for the first 3 weeks and CTLA4Ig (2 mg/kg) for additional 3 weeks.
Rats were pair fed, and body weight was checked daily. Systolic blood pressure (SBP) was evaluated at the end of the study using the tail-cuff method with a tail manometer tachometer system (BP-2000; Visitech System, Apex, NC). Pancreatic islet function was measured using IPGTT at 6 weeks. Before sacrifice, animals were housed in metabolic cages (Tecniplast Gazzada, Buguggiate, Italy) for 24 hours. The animals were anesthetized with intraperitoneal Zoletil 50 (10 mg/kg; Virbac Laboratories, Carros, France) and intraperitoneal xylazine (Rompun, 15 mg/kg; Bayer, Leverkusen, Germany). Pancreas tissue and blood samples were collected, and the pancreatic tissue was preserved as previously described.15,16 The serum creatinine (Scr) level was measured using an autoanalyzer (Coulter-STKS; Coulter Electronics, Hialeah, Finland). Trough level of TAC in whole blood was measured according to methods described previously.17
Evaluation of Pancreatic Islet Function and Viability
The area under the curve of glucose (AUCg) was determined for each subject using the trapezoidal method from the values obtained in the IPGTT. Insulin level in plasma was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Millipore Corporation, St. Charles, MO). Hemoglobin A1c (HbA1c) levels in the red cell lysates were determined by high-performance liquid chromatography (Bio-Rad, Richmond, CA). Ex vivo analysis was performed on pancreatic islets at the end of the experiment as described previously.18,19 Briefly, after collagenase digestion, the collected islets were placed in the wells of each group and were incubated for 1 hour in Roswell Park Memorial Institute (RPMI) 1640 medium (Wisent, Saint-Bruno, Quebec, Canada). The islets were washed with Krebs-Ringer Modified Buffer (KRB; 130 mmol/L NaCl, 3.6 mmol/L KCl, 1.5 mmol/L CaCl2, 0.5 mmol/L MgSO4, 0.5 mmol/L KH2PO4, 2.0 mmol/L NaHCO3, and 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and the supernatant was collected by centrifugation. The supernatant was collected (basal) and replaced with KRB containing 25 mM glucose for additional 30 minutes. The supernatant was sampled to measure the insulin level. Insulin concentration was measured using a rat insulin sandwich ELISA kit according to the manufacturer’s protocol (Millipore Corporation). Absorbance was measured at 450 nm using a VersaMax ELISA Microplate Reader (Molecular Devices, Sunnyvale, CA). Isolated islets were stained with 0.67 mmol/L acridine orange (AO) and 75 mmol/L propidium iodide (PI) (Sigma-Aldrich) to evaluate cell viability.19-21 The experiments were repeated at least 3 times.
Evaluation of the Pancreatic Beta Cell Area
We randomly selected 20 nonoverlapping islets per section for 9 animals in each group measured using a color image analyzer (TDI Scope Eye, version 3.0 for Windows; Olympus, Tokyo, Japan). Briefly, images were captured, and the sections were stained by immunohistochemistry for insulin and quantified using the Polygon program by measuring the percentage of insulin-positive cell areas per 0.5 mm2 of pancreas areas under 200× magnifications which included insulin-positive areas and excluded vacuoles. Histopathologic evaluation was performed on randomly selected areas of pancreatic tissue sections by a pathologist who was blinded to the sample group.
Immunohistochemistry was performed to identify oxidative stress markers, antioxidative stress-related molecules, and apoptosis using previously described methods.17 The 8-hydroxy-2'-deoxyguanosine (8-OHDG), an oxidative stress marker, and manganese superoxide dismutase (MnSOD) presence was assessed by incubating 4-μm tissue sections for 12 hours with an 8-OHDG antibody (JaICA, Shizuoka, Japan) and MnSOD antibody (Abcam, Cambridge, MA) at 4°C. The representative apoptotic marker, caspase-3, was detected by treating 4-μm tissue sections with an antibody specific for the active caspase-3 (Millipore, Billerica, MA) at 4°C for 12 hours. Apoptotic cell death was evaluated in the tissue sections using the In Situ Apoptosis Detection Kit (Millipore). The number of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end label (TUNEL)-positive cells was counted in 20 random areas in each section at 200× magnification. The procedure of ectodysplasin-1 (ED-1) (AbD Serotec, Oxford, UK) immunostaining was similar to that used for the staining of oxidative stress markers. The active cells of ED-1 were quantified in 20 random areas in each section at 200× magnification.
Evaluation of Serum 8-OHDG Level
Oxidative DNA stress was assessed by determining the expression of the DNA adduct 8-OHDG in serum samples using competitive ELISA (Cell Biolabs, San Diego, CA).
Transmission Electron Microscopy
Electron microscopic observation was performed as previously described.22 Minimum 20 random spots were scanned per section for the 9 animals of each group. The number of insulin granules per cell in the scanned areas was measured using an imaging analyzer (TDI Scope Eye, version 3.0 for Windows; Olympus).
In Vitro Effect of CTLA4Ig
To investigate the direct role of CTLA4Ig in TAC-induced pancreatic beta cell injury, we determined the effect of CTLA4Ig and TAC in insulin-secreting beta cell-derived line (INS-1) cells.23 We determined the apoptosis and oxidative stress in INS-1 cells. INS-1 cells were cultured in RPMI 1640 medium containing 11.1 mM sodium pyruvate, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10% fetal bovine serum (Wisent), 2 mM L-glutamine, 50 μM β-mercaptoethanol, 100 U/mL penicillin, and 100 mg/mL streptomycin (except for fetal bovine serum, all reagents were purchased from Sigma-Aldrich). The cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air for 24 hours, and subcultured to 70% to 80% confluence. After 24 hours, the medium was replaced with serum-free medium containing TAC (40 μg/mL) and CTLA4Ig (5 μg/mL), and the cells were incubated for 12 hours. All in vitro experiments were repeated at least 3 times, and experiments were performed using individual samples from separate experiments.
Measurement of Cell Viability
INS-1 cells were plated into 96-well plates at a density of 3 × 104 cells per well and preincubated for 24 hours at 37°C. The cell viability was determined using a Cell Counting Kit-8 assay (Dojin Laboratories, Kumamoto, Japan) according to the supplier’s instructions.24
Reactive oxygen species (ROS) production was assessed by flow cytometry. INS-1 cells were treated with serum-free medium containing the cell membrane (CM)-cell permeant, 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCFDA; Molecular Probes, Eugene, OR), according to the supplier’s protocol. Briefly, cells were plated into 12-well plates at a density of 2.5 × 105 cells/well and preincubated for 24 hours at 37°C. The cells were treated with serum-free medium containing 10 μM H2-DCFDA at 37°C for 1 hour. The cells were washed and detached in phosphate-buffered saline for further analysis by flow cytometry (BD Biosciences, San Diego, CA). For each analysis, 10 000 events were recorded.25,26
Data are presented as mean ± standard error (S.E.) of at least 3 independent experiments. Multiple comparisons among the groups were performed by 1-way analysis of variance with Bonferroni’s post hoc test using SPSS software (Software version 19.0; IBM, Armonk, NY). The results with P values less than 0.05 were considered significant.
Dose-Dependent Effect of CTLA4Ig on Pancreatic Islet Function and Cell Viability
Table 1 shows IPGTT results in the study groups. IPGTT in the CTLA4Ig groups did not differ from the VH group. However, groups of 1 and 2 mg/kg CTLA4Ig showed lower blood glucose level (at 30, 60, and 90 minutes) and AUCg than those of 0.25 and 4 mg/kg CTLA4Ig groups (Table 1). Thus, 1 and 2 mg/kg of CTLA4Ig were used in the conversion study. Figure 1 shows the results of the ratio of AO/PI staining and pancreatic islet function in the study groups. There was no difference in pancreatic islet viability and function between the CTLA4Ig and control groups, and there was no dose-dependent effect of CTLA4Ig on pancreatic islet viability, plasma insulin level, and glucose-stimulated insulin secretion (GSIS).
Effect of Conversion to CTLA4Ig on Metabolic Parameters in TAC-Induced DM
Figure 2 shows IPGTT and AUCg in rats with TAC treatment for 3 weeks. Fasting plasma glucose levels were not different between the control and TAC groups (75.7 ± 8.0 mg/dL vs 83.0 ± 8.0 mg/dL). However, the mean of the blood glucose levels at 30 minutes (475 ± 51 mg/dL), 60 minutes (360 ± 40 mg/dL), 90 minutes (288 ± 38 mg/dL), and 120 minutes (235 ± 35 mg/dL) in the TAC group was significantly higher than that of the control group (96 ± 6 mg/dL, P < 0.05). All rats in the TAC group satisfied the DM criteria (range, 204-336 mg/dL), and AUCg in the TAC group was approximately 2 times higher than that in the control group (279 ± 25 vs 155 ± 11 mg/dL per minute, P < 0.05).
After further 3 weeks, the rats of the TAC and TW groups had significantly increased body weight; however, conversion to CTLA4Ig (TC1 and TC2 groups) significantly reversed the change in body weight. Twenty-four-hour water intake, urinary volume, and Scr level significantly increased in the TAC group compared to the VH group. However, the groups converted to CTLA4Ig had decreased water intake, urinary excretion, and Scr level. The increase in HbA1c level decreased after TAC withdrawal or conversion to CTLA4Ig. SBP did not differ among the experimental groups (Table 2).
Effect of Conversion to CTLA4Ig on Pancreatic Islet Function in TAC-Induced DM
TW significantly decreased AUCg in the TW group compared to that of the TAC group, and, AUCg further decreased in TC1 and TC2 groups compared to the TW group (Figure 3, A and B; P < 0.05). TW significantly increased plasma insulin levels compared to the TAC group, and the plasma insulin levels recovered in TC1 and TC2 groups (Figure 3, C; P < 0.05). The insulin secretion capacity measured with GSIS decreased in the TAC group. However, TAC withdrawal significantly increased GSIS compared to the TAC group, and conversion to CTLA4Ig (in TC1 and TC2 groups) further improved insulin secretion compared to the TW group (Figure 3, D; P < 0.05).
Effect of Conversion to CTLA4Ig on Pancreatic Islet Size and Insulin Granules in TAC-Induced DM
Pancreatic beta cell area was measured using immunohistochemical staining for insulin. The TAC group had smaller islets and a lower expression of insulin staining within the islets (4.18 ± 0.27 μm2) than those of the VH group (11.82 ± 1.01 μm2) (Figures 4A and B; P < 0.05). TW reversed islet shrinkage induced by TAC (TAC, 4.18 ± 0.27 μm2 vs VH, 6.01 ± 0.37 μm2; P < 0.05) (Figures 4A and B), and conversion to CTLA4Ig produced a further recovery in islet size compared with the TW group (TW, 6.01 ± 0.37 μm2 vs TC1, 8.61 ± 0.46 μm2 and TC2, 8.78 ± 0.76 μm2; P < 0.05) (Figures 4A and B). Upon examination using electron microscopy, the number of insulin granules was markedly lower in the TAC group than VH group; however, increase in TW and conversion to CTLA4Ig further increased the number of insulin granules (Figure 5, A and B).
Effect of Conversion to CTLA4Ig on Oxidative Stress in Pancreatic Islet Cells in TAC-Induced DM
In this study, oxidative stress was evaluated using 8-OHDG detection. TAC significantly increased the 8-OHDG level in pancreatic islets and serum, and 8-OHDG level was decreased in the TW group and further decreased in the TC1 and TC2 groups (Figures 6A, B and D; P < 0.05). The activity of MnSOD decreased in the TAC group, but MnSOD expression recovered in the groups converted to CTLA4Ig (Figures 6A and C; P < 0.05).
Effect of Conversion to CTLA4Ig on Macrophage Infiltration in TAC-Induced DM
ED-1–positive cells were minimal in the VH group. The number of ED-1–positive cells was considerably higher in pancreatic sections from the TAC and TW groups than VH group. As expected, conversion to CTLA4Ig significantly decreased the number of ED-1–positive cells compared with the TAC and TW groups (Figures 7A and B; P < 0.05).
Effect of Conversion to CTLA4Ig on Apoptosis and Death of Pancreatic Islet Cells in TAC-Induced DM
The number of TUNEL-positive cells within the islets was significantly higher in the TAC group than in the VH group, and the number of TUNEL-positive cells was reduced in the TC1 and TC2 groups (Figures 8A and B; P < 0.05). Compared with the TAC group, TC1 and TC2 groups exhibited a decrease in the active form of caspase-3 in the islets (Figures 8A and C; P < 0.05). Islet viability was presented as the ratio of green fluorescence and red fluorescence in islet using AO/PI staining. The ratio of AO/PI was significantly reduced in the TAC group compared to the VH group (Figure 9A). However, TW increased the ratio, and groups converted to CTLA4Ig showed increased ratio as well, as shown in Figure 9B (P < 0.05).
Direct Effect of CTLA4Ig on TAC-Induced ROS Production and Cell Viability In Vitro
H2-DCFDA was used as a probe to detect the changes in intracellular ROS by flow cytometry. After the 12-hour treatment, 45.5% of TAC-treated cells stained positive for H2-DCFDA. Cotreatment with CTLA4Ig decreased the percentage of the H2-DCFDA–stained cell to 11.5%. CTLA4Ig treatment markedly reduced intracellular ROS production compared with TAC treatment alone (Figure 10, A and B; P < 0.05). To reveal the direct effects of CTLA4Ig, INS-1 cells were incubated with TAC with or without CTLA4Ig, and cell viability was evaluated by Cell Counting Kit-8. The cell viability of the cells was significantly greater with CTLA4Ig treatment compared to TAC treatment alone (Figure 10, C; P < 0.05).
The results of our study clearly demonstrated that CTLA4Ig was not diabetogenic and that the conversion to CTLA4Ig effectively controlled hyperglycemia in an experimental model of TAC-induced DM. Furthermore, the in vitro study revealed that CTLA4Ig reduced TAC-induced pancreatic islet injury. Previously, CTLA4Ig has been considered a possible replacement for CNI-induced allograft dysfunction. This study provided a rationale for conversion to CTLA4Ig in TAC-induced DM.
The majority of clinical studies on CTLA4Ig have shown that there was a lower rate of NODAT in patients treated with CTLA4Ig compared with those treated with CsA.27,28 This observation suggested that CTLA4Ig had a lower diabetogenic potential than that of CsA, but diabetogenicity of CTLA4Ig is still unknown. Therefore, we tested 5 escalating doses of CTLA4Ig (0.25-4 mg/kg) to determine whether CTLA4Ig had any diabetogenic effect and showed that none of the doses caused a significant increase in blood sugar measured by IPGTT compared with the VH group. In addition, plasma insulin level and insulin secretion in response to glucose loading were well preserved in CTLA4Ig-treated animals. Furthermore, CTLA4Ig did not affect pancreatic islet viability at any dose. These findings suggested that CTLA4Ig itself was not diabetogenic even at the highest dose used.
We designed a conversion study based on a clinical study that showed that CTLA4Ig conversion was effective in improving graft function in CsA-treated patients.27-29 We administered TAC for 3 weeks, and TAC treatment was continued, withdrawn, or converted to CTLA4Ig treatment for additional 3 weeks. This approach enabled us to evaluate the natural history of TAC withdrawal and the conversion effect of CTLA4Ig in TAC-induced DM. Before conversion to CTLA4Ig, we performed IPGTT to confirm development of TAC-induced DM and found that all rats satisfied the DM criteria. Interestingly, fasting plasma glucose levels in the TAC group were not significantly increased compared to the control group. This finding was consistent with a previous report that 25% to 30% of DM cases would not be detected if fasting plasma glucose was only used for diagnosis of NODAT,30 and it showed the importance of oral glucose tolerance test in diagnosis of NODAT.
Notably, TW improved pancreatic islet function (decreased blood glucose level, reduced AUCg, and improved plasma insulin level and GSIS). This finding suggested that TAC-induced DM could be reversible after TW. It is well known that impaired insulin secretion due to beta cell failure is the main mechanism of NODAT.28,29 Thus, the withdrawal of TAC is essential before irreversible change in TAC-induced pancreatic islet injury occurs and is promising in treating chronic TAC-induced pancreatic islet injury in clinical practice.
Treatment with CTLA4Ig further improved pancreatic islet function compared to the TW alone. We evaluated the effect of CTLA4Ig on TAC-induced oxidative stress, a common pathway of TAC-induced pancreatic islet injury.7,15,31-33 The results of this study showed that TAC-induced oxidative stress decreased with TW, and conversion to CTLA4Ig further decreased oxidative stress, inflammatory cell infiltration, and apoptotic cell death compared to TW treatment alone. We evaluated the direct effect of CTLA4Ig on TAC-induced pancreatic islet injury in vitro and found that administration of CTLA4Ig increased the cell viability and decreased ROS production in TAC-treated INS-1 cells. This finding suggested that CTLA4Ig may have an effect, improving islet cell function against TAC-induced pancreatic islet injury, and reduced oxidative stress may be responsible for the recovery from TAC-induced pancreatic islet injury observed when TAC-treated rats were converted to CTLA4Ig.
Kidney Disease: Improving Global Outcomes guideline recommends reduction, withdrawal, or replacement of CNIs with nondiabetogenic drugs in NODAT patients.34 The results of our study are applicable in clinical practice, but the availability of CTLA4Ig use is still limited. The reason for poor availability of CTLA4Ig may be shortage in the supply of belatacept35 and lack of data comparing TAC-based immunosuppression.36,37 Moreover, most clinical trials on CTLA4Ig in kidney transplant recipients have not included the study of pancreatic islet function.10,11 Thus, we expect that sufficient supply of belatacept and more clinical data of comparing TAC-based immunosuppression may expand belatacept use in kidney transplant recipients.
Our study has some limitations. First, we did not compare immunosuppression parameters of CTLA4Ig with those of TAC. Second, the 3-week treatment with CTLA4Ig after conversion was too short to translate it in exact treatment period in humans, but CTLA4Ig treatment for 3 weeks was carefully suggested in prospective of the “short” follow-up in rats. Third, we observed a direct recovery effect of CTLA4Ig on islet cell function against TAC-induced pancreatic islet injury, but the mechanism underlying this phenomenon is undetermined. Further studies are needed to define the immunological effects of CTL4Ig treatment compared with TAC treatment and to understand how CTLA4Ig improves TAC-induced pancreatic islet injury.
In conclusion, CTLA4Ig was not diabetogenic, and short-term conversion from TAC to CTLA4Ig treatment is effective in improving TAC-induced DM.
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