Simona Granata,Silvia Mercuri,Dario Troise,Loreto Gesualdo,Giovanni Stallone,Gianluigi Zaza
mTOR-inhibitors and post-transplant diabetes mellitus: a link still debated in kidney transplantation
post-transplant diabetes mellitus,kidney transplantation,immunosuppressive drugs,allograft co-morbidities,mTOR-inhibitor
The mammalian target of rapamycin inhibitors (mTOR-Is, Sirolimus, and Everolimus) are immunosuppressive drugs widely employed in kidney transplantation. Their main mechanism of action includes the inhibition of a serine/threonine kinase with a pivotal role in cellular metabolism and in various eukaryotic biological functions (including proteins and lipids synthesis, autophagy, cell survival, cytoskeleton organization, lipogenesis, and gluconeogenesis). Moreover, as well described, the inhibition of the mTOR pathway may also contribute to the development of the post-transplant diabetes mellitus (PTDM), a major clinical complication that may dramatically impact allograft survival (by accelerating the development of the chronic allograft damage) and increase the risk of severe systemic comorbidities. Several factors may contribute to this condition, but the reduction of the beta-cell mass, the impairment of the insulin secretion and resistance, and the induction of glucose intolerance may play a pivotal role. However, although the results of several in vitro and in animal models, the real impact of mTOR-Is on PTDM is still debated and the entire biological machinery is poorly recognized. Therefore, to better elucidate the impact of the mTOR-Is on the risk of PTDM in kidney transplant recipients and to potentially uncover future research topics (particularly for the clinical translational research), we decided to review the available literature evidence regarding this important clinical association. In our opinion, based on the published reports, we cannot draw any conclusion and PTDM remains a challenge. However, also in this case, the administration of the lowest possible dose of mTOR-I should also be recommended.
mTOR-inhibitors and post-transplant diabetes mellitus: a link still debated in kidney transplantation

The mammalian target of rapamycin inhibitors (mTOR-Is, Sirolimus, and Everolimus) are immunosuppressive drugs widely employed in kidney transplantation. Their main mechanism of action includes the inhibition of a serine/threonine kinase with a pivotal role in cellular metabolism and in various eukaryotic biological functions (including proteins and lipids synthesis, autophagy, cell survival, cytoskeleton organization, lipogenesis, and gluconeogenesis). Moreover, as well described, the inhibition of the mTOR pathway may also contribute to the development of the post-transplant diabetes mellitus (PTDM), a major clinical complication that may dramatically impact allograft survival (by accelerating the development of the chronic allograft damage) and increase the risk of severe systemic comorbidities. Several factors may contribute to this condition, but the reduction of the beta-cell mass, the impairment of the insulin secretion and resistance, and the induction of glucose intolerance may play a pivotal role. However, although the results of several in vitro and in animal models, the real impact of mTOR-Is on PTDM is still debated and the entire biological machinery is poorly recognized. Therefore, to better elucidate the impact of the mTOR-Is on the risk of PTDM in kidney transplant recipients and to potentially uncover future research topics (particularly for the clinical translational research), we decided to review the available literature evidence regarding this important clinical association. In our opinion, based on the published reports, we cannot draw any conclusion and PTDM remains a challenge. However, also in this case, the administration of the lowest possible dose of mTOR-I should also be recommended.

New-onset diabetes after transplantation (NODAT) refers to the occurrence of diabetes in previously non-diabetic clinically stable kidney transplant recipients who had been discharged from the hospital and tapered to their maintenance immunosuppressive therapy (1). It occurs in almost 4% to 27% of kidney transplant recipients (2–6) and it may induce the development of the chronic allograft damage by activating several pro-fibrotic mediators [including transforming growth factor beta (TGF-β)] and promoting mesangial matrix expansion and cell hyperplasia (7), accelerating the onset of severe clinical complications/comorbidities. To diagnose NODAT, the International Consensus Guidelines published in 2003 recommended to use the same diagnostic criteria adopted by the American Diabetes Association and the World Health Organization (ADA/WHO) for type 2 diabetes in non-transplant patients: fasting glucose ≥126 mg/dL (7 mmol/L) on more than one occasion, random glucose ≥ 200 mg/dL (11.1 mmol/L) with symptoms, two-hour glucose after 75 g oral glucose tolerance test of ≥200 mg/dL (11.1 mmol/L), and hemoglobin A1C (HbA1c) ≥ 6.5% (3). However, since the difficulty to screen all patients before transplantation and to identify pre-existing forms of diabetes, in 2013, a second international consensus changed the nomenclature from NODAT to post-transplantation diabetes mellitus (PTDM; a more inclusive term for diabetes that is diagnosed after transplantation without specific reference to any previous history of diabetes) (3). Risk factors for PTDM are similar to those for type 2 diabetes mellitus: increased age (>40 years), family history of type 2 diabetes, ethnicity (African-American, Asian, and Hispanic patients are at higher risk compared to Caucasian), abnormal glucose tolerance (expressed by fasting blood sugar levels between 90 and 100 mg/dL), and specific genetic factors (8–12). Other risk factors for PTDM include the metabolic syndrome and the obesity, responsible for the insulin resistance and the glucose intolerance. Indeed, the adipose tissue from obese subjects increases the expression of Tumor Necrosis Factor-α (TNF-α) (13), which downregulates genes involved in the insulin action (such as glucose transporter (GLUT)-4, insulin receptor, and insulin receptor substrate (IRS)-1) (14, 15), reduces adiponectin release and stimulates adipocyte lipolysis (16). Also, viral infections may increase the risk of PTDM. Hepatitis C virus (HCV) (17) infection may trigger an immune-mediated reaction against β cells with consequent cytopathic effects, glucose uptake reduction (18), and gluconeogenesis augmentation (19). Likewise, cytomegalovirus (CMV) may directly damage beta-cells by the upregulation of large pro-apoptotic machinery or, indirectly, by facilitating the release of pro-inflammatory cytokines (20). However, in kidney transplant recipients, the administration of immunosuppressive drugs may further impact the incidence of PTDM. Corticosteroids (administered at high dosages as part of the induction therapy and/or for treating acute rejection and as main constituent of the maintenance immunosuppressive protocol) and calcineurin inhibitors (CNIs; Tacrolimus and Cyclosporine A) are the main responsible for this complication, while the diabetogenic effects of mammalian target of rapamycin inhibitors (mTOR-Is) are still debated (21).

mTOR inhibitors (mTOR-Is, Sirolimus, and Everolimus) exert their pharmacological effects via inhibition of the serine/threonine kinase mammalian target of rapamycin (mTOR). mTOR integrates signals from growth factors, hormones, nutrients, and cellular energy levels to regulate protein translational and cell growth, proliferation, and survival (22). mTOR exists in two complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (23). mTORC1 also includes RAPTOR (24, 25), MLST8 (26), PRAS40 (27), and DEPTOR, while mTORC2 includes RICTOR, MAPKAP1, PRR5/PRR5L, Mlst8, and Deptor (28–30). This complex is less sensitive to the acute treatment with mTOR-Is, while long-term administration of these medications inhibits mTORC2 activity by acting on complex integrity (31, 32). The use of mTOR-Is as immunosuppressants in solid organ transplantation is mostly based on their ability to block cell cycle progression from G1 to the S phase and cellular proliferation (33). Additionally, these drugs may have further therapeutic effects by modifying: (a) protein synthesis, (b) cell cycle, (c) lipid metabolism, (d) energy metabolism, (e) autophagy, (f) angiogenesis, (g) cytoskeleton remodeling, and (h) epithelial to mesenchymal transition (23, 34–39). Because of their specific pharmacological characteristics, mTOR-Is are highly effective in renal transplantation, and owing to their relative lack of nephrotoxicity, these inhibitors are a valid alternative to CNIs for the maintenance of immunosuppression in renal transplant recipients with chronic allograft nephropathy (40). Although the clinical utility of this drug category is clear, as other immunosuppressive drugs, mTOR-Is may persuade the development of some adverse effects that need to be immediately recognized and treated to avoid severe illness in kidney transplant recipients. Numerous clinical and translational studies have highlighted that mTOR-Is-treated kidney transplant recipients may develop metabolic disorders, including PTDM (41–43). As proven in in vitro and in animal models, mTOR-Is may decrease beta-cell mass through an increment of the rate of apoptosis (44–47), induce impairment of glucose-induced insulin secretion (45–49), and facilitate glucose intolerance and insulin resistance (50–52) (Table 1). The impact of mTOR-Is on glucose metabolism is also mediated by its interference with insulin signal transduction. Physiologically, insulin and insulin-like growth factors (IGF) activate mTORC1 through the IRS/PI3K/Akt pathway (50). The mTOR-Is bind to mTOR and suppress the PI3K/AKT pathway (23). The reduction of Akt phosphorylation causes an increment in beta-cell apoptosis with consequent reduced beta-cell mass, impairment of glucose-stimulated insulin secretion, and proinsulin biosynthesis (48). This leads to augmented gluconeogenesis (58), reduced glucose uptake (54), glycolysis, and glycogen synthesis (41, 59) (Table 1). However, these properties seem to depend on the metabolic context in which they are studied. mTOR-I administered to an animal model of nutrition-dependent type 2 diabetes (diabetic Psammomys obesus) worsened the metabolic state of the diabetic animals: augmented insulin resistance, β-cell dysfunction, and death, thereby preventing β-cell adaptation to hyperglycemia (41). Hyperglycemia and glucose intolerance after mTOR-Is treatment is due to the upregulation of several gluconeogenic genes in the liver via the coordinated activation of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha, cAMP response element-binding protein (CREB), CREB-regulated transcription coactivator 2 (CRTC2), and forkhead box O1 (FoxO1) (58). Reduced obesity and hyperlipidemia are frequently associated with the mTOR-I treatment (58, 60). Both in vitro and in vivo studies have revealed the association between adipogenesis and the mTOR pathway (61–64). Notably, obesity and overnutrition trigger chronic hyperactivation of mTOR activity in multiple tissues (50, 61). In humans, increased S6K activity (65, 66) and overphosphorylation of translation suppressor 4EBP have been observed in obesity (67). Furthermore, accelerated adipogenesis and obesity have been reported in mice lacking 4E-BP1 and 4E-BP2 genes (68). Indeed, targeting the mTOR pathway has been suggested as a treatment for obesity. Consistently, S6K knockout mice were protected against obesity due to increased lipolysis levels and energy expenditure (61, 69, 70). mTOR-Is treatment may, then, reduce the cell number in adipose tissue with a small contribution from reduced adipocyte size. Reduced lipid uptake and fat cell number impairs the capacity of adipose tissue for plasma lipid clearance, which likely contributes to hyperlipidemia (58). The inhibition of the mTOR pathway may also play a role in insulin secretion in pancreatic β-cells. Several studies have reported the inhibition of glucose-induced insulin secretion in clonal β-cell lines and in islets (41), but the exact mechanism is unclear (44). The control of insulin secretion in beta-cells involves several signaling pathways. One proposed mechanism is that inhibition of mTORC1 decreases mitochondrial function, specifically, the activity of α-ketoglutarate dehydrogenase. This results in reduced carbohydrate metabolism and therefore, reduced mitochondrial ATP production (55), which regulates insulin secretion in β-cells (71). Other explanation that rapamycin promotes autophagy, and the intracellular degradation of cytoplasmic proteins involved in the insulin production and secretion (56). Moreover, several in vitro studies have reported a leading role of mTORC2 in the function and survival of beta-cells (46, 72) and insulin resistance (53). Chronic treatment with mTOR-I also inactivates mTORC2 with subsequent inhibition of AKT signaling (46) and other mTORC2 substrates such as protein kinases PKCα, SGK1 substrate NDRG1 in the liver, muscle, and white adipose tissue (57, 73). It has also been observed that long-term treatment with mTOR-Is (20 weeks) partially restored the detrimental effects on metabolism with enhanced insulin sensitivity, increased oxygen consumption, and improved serum lipid profile with a certain degree of glucose intolerance (74). Furthermore, in several maintenance immunosuppressive therapeutic protocols, mTOR-I are combined with CNIs. Numerous observations have reported that CNIs treatment may lead to PTDM by a multifactorial mechanism, which includes impaired insulin secretion, insulin resistance, altered glucokinase function, mitochondrial impairment, and pancreatic β-cell apoptosis (75–79). The mechanism in pancreatic insulin-secreting β cells seems to be mediated by the inhibition of two targets of calcineurin: nuclear factor of activated T-Lymphocytes (NFAT) and cAMP responsive element binding protein (CREB) (80). Both transcription factors mediate the expression of IRS-2 which promotes β-pancreatic cell growth, proliferation, and survival, insulin secretion by mediating phosphorylation of Akt in response to insulin and insulin-like growth factor (IGF)-1 (81). Through the inhibition of these signaling pathways, CNIs diminish β-cell survival and replication and promote β-cell failure (81–84). Additionally, pancreatic islets treated with CNIs showed significant morphological alterations in the form of cytoplasmic swelling and vacuolization, degranulation, and immunohistochemical and ultrastructural loss of secretory granules (85, 86). Tacrolimus (TAC) appears also to reduce insulin secretion through a downregulation of the production of ATP and glycolysis due to a reduced activity of glucokinase, a rate-limiting enzyme in glycolysis that represents an important glucose sensor in pancreatic β-cells (78). The diabetogenic effect of TAC can also be enhanced by mitochondrial dysfunction through a decrease in both mitochondrial respiration activity and mitochondrial mass (87). Most studies comparing the diabetogenic effects of the CNIs report higher rates of PTDM among patients receiving TAC compared to cyclosporine A (CsA) (88–90).

During the last two decades, mTOR-Is (mainly Everolimus) have been widely used as part of the maintenance immunosuppressive therapy of kidney transplant recipients and clinical studies/trials have investigated their possible pathogenetic impact on PTDM (Table 2). As reported by the SYMPHONY study after 1 year and 3 years of follow-up (91, 92), patients treated with low-dose sirolimus (SRL) plus Mycophenolate Mofetil (MMF) presented a higher incidence of PTDM than those treated with low dose of CsA plus MMF (6.6% vs. 4.2% after the first year and 8% vs. 5% after 3 years of follow-up). No differences were observed in the comparison between the standard dose of CsA vs. low dose SRL (in both groups the incidence was 8%). This study suggested that a low-dose CsA-based maintenance immunosuppressive treatment or a switch from TAC to low dose of CsA or SRL could be beneficial for kidney transplant recipients at high risk of PTDM. However, in our opinion, this therapeutic strategy should be undertaken only in highly selected patients after weighing the risk of rejection or in the absence of additional adverse events. In the prospective randomized SPIESSER study, which compared the safety and efficacy of a SRL plus MMF-based immunosuppressive regimen with a CsA plus MMF-based regimen after an induction therapy with polyclonal antilymphocyte antibodies and withdrawal of steroids at 6 months’ post-transplantation, it was observed a higher incidence of PTDM in the SRL group in the first-year post-transplantation (9% vs. 3%, p = 0.07) (93). Instead, no differences were reported between the 2 study groups after 5 years of follow-up (2% vs. 4%, p = 0.69) (94). This study revealed potential diabetogenic effects of the mTOR-Is in the early post-transplant phase (probably induced by the high-dosages of these drugs in association with corticosteroids). However, the relatively low number of patients with a diagnosis of PTDM, the similar risk of PTDM between the two study groups, and the high rate of conversion from the randomized immunosuppression to other regimens in the SRL group may not allow reaching a definitive conclusion revealing the need of a larger trial on this specific topic. Kreis et al. (ORION Study) also described no differences in the incidence of hyperglycemia and insulin-dependent PTDM in patients treated with SRL compared to CsA (95). The evaluation of two SRL-based regimens, one with CNI withdrawal (SRL + TAC-Elim) and the other with complete CNI avoidance (SRL + MMF), compared with a CNI-based regimen containing TAC + MMF in de novo renal allograft recipients demonstrated higher incidence of PTDM in TAC recipients confirming a diabetogenic effect of TAC compared to SRL (96). These results could be partially explained by the relatively high trough level of TAC used in this group. Also in the conversion trials, the switch from CNI to mTOR-I has not shown clear diabetogenic effects. However, some authors have reported an increased risk of PTDM in mTOR-I-treated patients due to a drug-related enhancement of peripheral insulin resistance and impairment of the compensatory beta-cell response (97). In both CONCEPT and ZEUS studies, the early conversion from CsA-based to SRL-based therapeutic regimen (3 or 4.5 months after transplantation) did not induce PTDM in a 12 months-period post-transplantation (98, 99). Similarly, in the late conversion (CONVERT) study, where the renal allograft recipients were randomly assigned (2:1) to undergo conversion from CsA- or TAC-based immunosuppression to SRL or to continue receiving CNI-based therapy for 2 years, the frequency of PTDM was similar between the two study regimens (4.7% vs. 4.4%, p = 1.000) (100). Holdaas et al., in the ASCERTAIN study, which included kidney transplant patients with allograft impairment (GFR 30–70 mL/min) who underwent the minimization of CNI or conversion to Everolimus (EVR), reported a percentage of patients with PTDM of 4.7% in the CNI withdrawal group, 4.9% in the minimization group and 3.3% in the control group (101). All these studies (98–101), although performed using SRL alone in a heterogeneous patients’ population, encourage clinicians to reduce the dosages of this immunosuppressive drug. Nevertheless, a recent systematic review and meta-analysis established that the conversion from CNIs to mTOR-Is did not significantly decrease the risk of PTDM (111). Instead, Veroux et al. (102) showed that the conversion from CNI to mTOR-I-based therapy in patients with PTDM had a positive effect on insulin-stimulated glucose uptake. In this study, it was observed an improved glucose balance in 80% of patients converted to SRL compared with those patients (37.5%) in whom a reduction in CNI dose was carried out. No change was found in the incidence of acute rejection. According to these authors, such beneficial effects of mTOR-Is on the glycemic homeostasis could be explained by the chronic inhibition of mTORC1 (a biological/pharmacological effect such as that observed after metformin administration) (112). In this single-center study, the small sample size (particularly of patients treated with mTOR-Is) and the low incidence of PTDM (probably due to an early reduction of immunosuppression) cannot allow to draw any definitive conclusions. In all cases, a conversion from CNIs to mTOR-Is should be achieved only after a carefully benefit–risk evaluation. Unfortunately, also the impact of the combined therapy of mTOR-Is plus CNIs on PTDM is still argued. Vitko et al. in a 36-month, multicenter, randomized, parallel-group equivalence trial of two oral doses of EVR (1.5 or 3 mg/day) vs. MMF (2 g/day) along with CsA microemulsion (Neoral) and corticosteroids in de novo renal transplant recipients, reported a higher incidence of PTDM in patients receiving 3 mg/day EVR (12.6%) compared to those receiving a low dose of EVR (6.7%) and MMF (5.6%) (103). In another 6-month, randomized, open-label, parallel-group, comparative trial comparing two regimens of TAC plus SRL (with either 0.5 or 2 mg) with a TAC plus MMF immunosuppressive schema, authors found that a larger number of patients treated with TAC plus SRL at 2 mg developed PTDM (p = 0.005). However, the number of patients requiring insulin for PTDM was similar in the TAC/SRL 2 mg and TAC/MMF groups (p > 0.05) (104). In a multicenter trial, in which renal transplant recipients were randomized to TAC with fixed-dose SRL (N = 318) or TAC with MMF (N = 316), 6 months’ creatinine clearance was comparable between the 2 immunosuppressive schemas. Biopsy-confirmed acute rejection was 15.1% (TAC/SRL) and 12.3% (TAC/MMF). In both groups, graft survival was 93% and patient survival was 99%. Premature withdrawal due to an adverse event was twice as high in the TAC/SRL group (15.1% vs. 6.3%). The incidence of any antidiabetic treatment for >30 consecutive days in previously nondiabetic patients was 17.8% in TAC/SRL, and 24.8% in TAC/MMF (105). Guerra et al., have, then, studied a long-term follow-up post-transplant (8 years) to compare TAC/SRL, TAC/MMF, and CsA/SRL. In this report, the incidence of PTDM was not significantly different among the three groups (p = 0.37), while a slightly smaller percentage of PTDM was registered in the TAC/MMF group (19%) than TAC/SRL (32%) and CsA/SRL (31%) group. However, difference did not reach any statistical difference (p = 0.16) (106). These results were confirmed by Gallon et al. and by Chhabra et al. (107, 108). Moreover, in a 24-month, open-label study, 833 de novo renal-transplant recipients were randomized to EVR 1.5 or 3 mg/day (target troughs 3–8 and 6–12 ng/mL, respectively) with reduced-exposure CsA, or mycophenolic acid (MPA) 1.44 g/day plus standard-exposure CsA. The overall incidence of PTDM and adverse events were comparable between the groups. Corticosteroids were used in more than 99% of patients in each group during the study, with more than 70% receiving corticosteroids without discontinuation throughout the 24-month study period (109). These studies (104–109) demonstrated a similar impact of the combined therapy of CNIs and mTOR-Is on PTDM. Johnston et al. demonstrated that the incidence of PTDM was 21.9% in patients treated with a combination of SRL plus CsA, 21.5% in those treated with SRL plus TAC, and 17.8% in the group of patients received SRL plus MMF/AZA, showing that rapamycin was an independent variable involved in the development of PTDM (42). This study, although performed on a large dataset, has major limitations including the inherent limitations of retrospective analyses of administrative data sets, the absence of information regarding the dosage of SRL and CNI used, the enrolment of patients who had Medicare as the primary payer, which may limit the applicability of its findings to other patient populations. In the recent TRANSFORM (Transplant efficacy and safety outcomes with an EVR-based regimen) study, a 24-month, prospective, open-label trial in 2037 de novo renal transplant recipients randomized (1:1) within 24 h of transplantation to receive EVR with reduced-exposure CNI (EVR + rCNI) or mycophenolate with standard-exposure CNI, the incidence of PTDM was similar in both the arms (19.6% vs. 18.6%) (110). Even if PTDM was not included as a primary endpoint, this study confirmed the no specific diabetogenic effects of the combined therapy with CNIs plus mTOR-Is (particularly administered at low dosages), as previously suggested. Finally, a recent network meta-analysis involving 206 eligible studies that identified 75,595 patients on TAC, 51,242 on CsA, and 3,020 on SRL, demonstrated that TAC tended to exhibit higher diabetogenicity in the short-term (2–3 years post-transplant), whereas SRL exhibits higher diabetogenicity in the long-term (5–10 years post-transplant) (113). This study is quite difficult to interpreter due to the clinical heterogeneity of the immunosuppression protocols utilized in the included studies (such as co-treatments and therapy used for rejections), variability of the criteria used to define PTDM, and absence of control of several clinical/therapeutic confounding factors.

After reviewing the available literature on this topic area, we cannot draw any definite conclusions about the diabetogenic impact of the mTOR-Is. However, we can encourage clinicians to lower the dose of these immunosuppressive drugs in patients at high risk of PTDM. Moreover, our paper shows that the transplant scientific community should undertake more research programs to better study this important topic.

SG, SM, DT, and GZ searched the literature and wrote the manuscript. GZ, GS, and LG revised the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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