1 Division of Nephrology, Department of Medicine, 2 Department of Surgery and 3 Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, NY, USA
Correspondence and offprint requests to: Glen S. Markowitz, MD, Department of Pathology, Columbia University Medical Center, 630 West 168th Street, Room 14-224, New York, NY 10032, USA. Email: gsm17{at}columbia.edu
Keywords: calcineurin inhibitor; de novo thrombotic microangiopathy; renal transplantation; sirolimus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Treatment with calcineurin inhibitors (CNIs) is a well-established risk factor for the development of de novo TMA. A recent, large analysis of the United States Renal Data System (USRDS) and Medicare claims identified multiple additional risk factors including younger recipient age, older donor age, female gender of the recipient, longer duration of dialysis before transplantation, previous renal transplant, delayed graft function (DGF), allograft rejection, increased peak panel-reactive antibody, and treatment with sirolimus (SRL) [2]. Anti-cardiolipin antibody seropositivity, often in the setting of chronic hepatitis C virus infection, is an additional risk factor for de novo TMA [3].
SRL (rapamycin) is an immunosuppressive agent commonly administered to renal transplant recipients. SRL may be used in combination with a CNI or as an alternative agent. A recent, large trial has shown that SRL facilitates early CNI withdrawal and that this regimen is associated with less long-term nephrotoxicity [4]. The most frequently reported side effects of SRL are thrombocytopenia, leukopenia, hypertriglyceridaemia and hypercholesterolaemia [5].
SRL has a similar mechanism of action to the CNIs. Cyclosporin (CSA), tacrolimus (TAC) and SRL all produce their immunosuppressive effect by binding to cytoplasmic proteins called immunophilins that modify immune function. CSA binds cyclophilin, and TAC binds FK-binding protein 12 (FKBP12); these complexes in turn inhibit calcineurin, a calcium-dependent phosphatase required for interleukin-2 production and progression of T cells from the G0 to G1 phase of the cell cycle. SRL also binds FKBP12, but the complex does not inhibit calcineurin activity. Instead, the SRLFKBP12 complex inhibits a cell cycle regulatory protein referred to as mammalian target of rapamycin (mTOR) [6]. Inhibition of mTOR blocks the cellular response to cytokines and growth factors and progression of T cells from the G1 to S phase of the cell cycle. Inhibition of mTOR by the FKBPSRL complex also has been shown to block proliferation in non-lymphoid tissues, including hepatocytes, vascular smooth muscle cells, endothelial cells and renal tubular epithelial cells [7].
Although initial studies suggested that SRL lacked significant nephrotoxicity, recent reports have challenged this concept. Early SRL use is associated with prolongation of DGF and a distinct histological pattern of renal injury described as a cast nephropathy in which distal tubular casts are associated with sloughed tubular epithelia [8]. Based on an animal model of ischaemic acute tubular necrosis, the mechanism of SRL-induced DGF probably involves increased tubular apoptosis and reduced cellular proliferation, mediated in part by inhibition of protein kinase p70S6K [7].
Recent evidence suggests that treatment with SRL may be followed by the development of TMA. First, TMA has been noted in patients on protocols containing SRL in conjunction with CNIs [5,910]. Secondly, a recent analysis of the USRDS identified SRL use post-transplantation as a risk factor for TMA [2]. Thirdly, a recent report described a patient who developed TMA 16 days after renal transplantation on an immunosuppression regimen containing SRL, mycophenolate mofetil (MMF) and prednisone, after thymoglobulin induction [11]. This is the only report of SRL-associated TMA in the absence of a CNI. We present two additional cases of biopsy-proven TMA in patients treated with SRL who were not receiving CNIs at the time of renal biopsy.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Of note, SRL was introduced at Columbia University Medical Center on a compassionate use basis in 1999 and for routine use in 2000. Both patients provided consent for the preparation of this manuscript.
![]() |
Case 1 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Renal allograft biopsy findings
Sampling for light microscopy included two cores of renal cortex and 15 glomeruli, two of which were globally sclerotic. Two glomeruli contained intracapillary fibrin thrombi (Figure 1A) and a few additional glomeruli exhibited ischaemic-type wrinkling of the glomerular basement membrane and mesangiolysis. The remaining glomeruli were unremarkable. Proximal tubules displayed mild degenerative changes characterized by luminal ectasia and cytoplasmic simplification. There was mild tubular atrophy and interstitial fibrosis involving 15% of the cortex sampled. Mild interstitial inflammation composed mainly of lymphocytes occupied <20% of the cortex sampled. There was no significant tubulitis or neutrophil margination in peritubular capillaries. A single artery contained a fibrin thrombus that was associated with endothelial necrosis (Figure 1B). Multiple vessels displayed mucointimal oedema (Figure 1C).
|
Based on these findings, the patient was diagnosed with acute TMA.
Clinical follow-up
Following renal biopsy, treatment with SRL was discontinued and replaced with TAC 4 mg twice per day. Prednisone and MMF were continued. CMV polymerase chain reaction (PCR) viral loads and anti-cardiolipin antibody were negative. Within 2 weeks, the patient's creatinine declined to 2.1 mg/dl and her platelet count increased to 191 000/mm3. Six months later, the patient has a serum creatinine of 1.8 mg/dl, a haematocrit of 36% and a platelet count of 237 000/mm3.
![]() |
Case 2 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemical staining for C4d was negative in peritubular capillaries, providing evidence against antibody-mediated rejection. Immunofluorescence and electron microscopy were not performed.
Based on these findings, three diagnoses were rendered: (i) acute TMA; (ii) moderate acute rejection (Banff grade 2A); and (iii) moderate chronic allograft nephropathy.
Clinical follow-up
The patient was treated for acute rejection with intravenous methylprednisolone 500 mg daily for 3 days followed by prednisone taper. The SRL dose was decreased to 5 mg once per day, and his SRL trough level decreased to 11.1 ng/ml. His creatinine decreased to 2.2 mg/dl, but 1 month later climbed again to 3.6 mg/dl. His platelet count remained depressed at 88 000/mm3. Examination of a peripheral blood smear revealed rare schistocytes. A fourth renal biopsy showed (i) findings of TMA, which now appeared more subacute to chronic; (ii) moderate to severe chronic allograft nephropathy; (iii) no evidence of acute rejection; and (iv) negative staining of peritubular capillaries for C4d.
SRL was discontinued and the patient was started on prednisone 80 mg per day and plasma exchange. Despite 18 plasma exchange treatments, his platelet count remained low at 70 000/mm3. Six weeks later, the patient had a creatinine of 2.7 mg/dl. The patient was started on vincristine and, following a single dose, was admitted with catheter-related infection. Plasma exchange was discontinued and the patient was treated with intravenous immunoglobulin (IVIG). Three days later, he developed acute renal failure with a creatinine of 8.3 mg/dl. A fifth renal allograft biopsy revealed chronic sequelae of the previous findings and superimposed acute tubular injury due to osmotic nephrosis, consistent with IVIG nephrotoxicity.
Treatment with IVIG was discontinued. The patient's creatinine declined over 1 month to a new baseline level of 3.0 mg/dl. The patient subsequently progressed to ESRD over the following 8 months. During this time, his immunosuppressive regimen consisted of azathioprine 25 mg once per day and prednisone 10 mg once per day. Two years later, at the time of his fourth renal transplant, allograft nephrectomy revealed prominent changes of chronic thrombotic microangiopathy. At that time, enzyme-linked immunosorbent assay (ELISA) testing did not reveal antibodies against the spouse's (donor's) HLA antigens.
Results
The clinical parameters of the two patients with de novo TMA following treatment with SRL are summarized in Table 1. Both patients had risk factors for development of de novo TMA including, in case 1, female gender, prolonged duration of dialysis pre-transplantation, and DGF, and, in case 2, previous transplantation and previous allograft rejection. Both patients underwent renal allograft biopsy due to an elevation of serum creatinine (rather than systemic symptoms of TMA) and both were diagnosed with TMA based on biopsy findings. The duration of SRL therapy prior to biopsy was relatively short at 4.5 and 2 weeks, respectively. The histological findings of TMA were accompanied by thrombocytopenia, a known side effect of treatment with SRL. Although neither patient had an acute decline in haematocrit, patient 1 had a mild increase in LDH and patient 2 had rare schistocytes on peripheral smear.
|
Discussion
We report the clinical and pathological findings in two patients who developed de novo TMA following treatment with SRL. In case 1, the rapid and sustained improvement in renal function following discontinuation of SRL strongly implicates this agent in the development of acute TMA. Case 2 is more complex in that the patient had previous and concurrent episodes of acute cellular rejection and a history of CNI toxicity. For this same reason, this case is also of particular interest. The patient had two recent previous renal allogaft biopsies documenting CNI toxicity (following treatment with TAC and CSA), but neither biopsy exhibited findings of TMA. The close temporal relationship between the onset of treatment with SRL and the development of TMA strongly implicates SRL as the aetiological agent. Unfortunately, due to multiple co-existent processes, SRL withdrawal did not lead to improvement of renal function.
Multiple previous reports have suggested a relationship between SRL and the development of TMA. Barone et al. described a patient who developed TMA 16 days after renal transplantation on an immunosuppression regimen containing SRL, MMF and prednisone, after thymoglobulin induction [11]. Robson et al. reported a patient with stable renal function on CSA for 8 years. Due to mild chronic allograft nephropathy and leukopenia, azathioprine was discontinued and replaced with SRL. Shortly thereafter, the patient developed acute TMA [9]. Similarly, Saikali et al. described a patient with CNI toxicity related to treatment with TAC [10]. Following introduction of SRL and a marked reduction in TAC dose, the patient developed TMA. Treatment with TAC was discontinued but the patient continued to have worsening TMA. Only following discontinuation of SRL and plasma exchange did the patient's TMA resolve and renal function improve [10]. In a large, phase III study comparing SRL with placebo in renal transplant recipients simultaneously treated with CSA and steroids, TMA was among the most frequent diagnoses leading to SRL discontinuation [5]. SRL has also been linked to acute TMA in patients with steroid-refractory graft-versus-host disease following allogeneic haematopoietic stem cell transplantation [12].
A recent study looked at risk factors for TMA in 15 870 renal transplants recipients in the USRDS [2]. The diagnosis of TMA was based on diagnostic coding at the time of hospital discharge. Based on the design of the study, the data must be interpreted with caution. For instance, a patient might have received SRL because of the fear of using a CNI in a patient with a previous history of TMA. Furthermore, a patient may have been discharged while receiving SRL due to previous CSA-induced TMA, followed by SRL rescue. Despite these potential issues, the results in this large cohort were quite striking. The incidence of TMA among patients on initial SRL maintenance therapy was 18.1 in 1000 patient-years (PY), compared with only 5.0 in 1000 PY among patients on initial maintenance CNI therapy. On univariate analysis, SRL therapy, with or without concurrent CNI, was associated with de novo TMA.
Calcineurin inhibitors have multiple effects on the renal vasculature and are established precipitants of TMA in the renal transplant recipient. CNIs are associated with direct endothelial toxicity, as well as vasoconstriction and hyaline arteriolopathy [13]. In cell culture, CSA has an anti-proliferative and pro-apoptotic effect on endothelial cells [14] and blocks angiogenesis induced by vascular endothelial growth factor [15]. In vivo, higher CSA trough levels correlate with elevated levels of circulating endothelial cells, a marker of endothelial damage [16]. CSA also blocks repopulation of the allograft vasculature by recipient-derived endothelial cells [17].
SRL may promote thrombosis and TMA by a mechanism similar to that of the CNIs. The SRLFKBP12 complex inhibits mTOR, which in turn prevents p70S6K activation. Endothelial cell proliferation in response to growth factors and oscillatory blood flow is highly dependent on p70S6K signalling [18,19]. Thus, similar to the CNIs, SRL acts to inhibit endothelial cell proliferation [7], a property that is the basis for the use of SRL-eluting coronary artery stents to prevent restenosis [20]. In the transplant setting, endothelial cells may be injured by a variety of mechanisms including direct drug toxicity, infection, immune processes and OKT3 use. In response to injury, blood vessels in the allograft may be repopulated by recipient-derived endothelial cells [21]. The anti-proliferative effect of SRL and the CNIs may prevent repopulation of the allograft vasculature by reparative endothelial proliferation, thereby promoting local activation of the clotting cascade, consumption of platelets and red blood cell destruction. This hypothesis is supported by the fact that the majority of de novo TMA represents renal-limited rather than systemic disease. SRL has also been shown in cell culture to promote platelet aggregation [22], thus providing another potential mechanism by which SRL could promote TMA.
How can SRL produce TMA when it has been used with success in patients with de novo TMA associated with CNI use [23,24]? This apparent paradox may be explained by SRL's different mechanism of action and the need for specific CNI withdrawal to control TMA, just as TMA associated with CSA may respond solely to switching to TAC [25]. In a similar manner, it is worth noting that in the case reported by Barone et al. [11] and the first case reported herein, de novo TMA improved following discontinuation of SRL and institution of a CNI.
Thrombocytopenia is a well-recognized side effect of SRL, which is dose dependent and rapidly reversible following cessation of SRL therapy [26]. Because of this side effect, one of the main clinical parameters that may suggest the presence of TMA is of limited utility in patients treated with SRL. As a result, TMA in patients treated with this agent may be under-recognized and requires extra diligence on the part of the clinician. Renal biopsy is likely to remain the most useful modality to identify TMA in patients treated with SRL.
SRL has been a useful advance in the field of transplantation. Initial reports that this agent was not associated with significant nephrotoxicity have proven false, although the incidence of renal side effects is far lower than that of the CNIs. We report two patients with de novo TMA following treatment with SRL. These cases add to a growing body of literature that points to an aetiological relationship between SRL and the development of TMA, with or without co-administration of a CNI. Additional studies are needed to compare the thrombogenic potential of SRL and the CNIs and to determine whether these effects are additive when the agents are administered together.
Conflict of interest statement. R. John Crew is supported by a grant from the Kidney and Urology Foundation of America.
[See related article by Marti and Frey (this issue pp. 1315)]
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|