Calcineurin-inhibitor therapy can lead to renal dysfunction in heart transplantation patients. The novel immunosuppressive (IS) drug sirolmus (Srl) lacks nephrotoxic effects; however, proteinuria associated with Srl has been reported following renal transplantation. In cardiac transplantation, the incidence of proteinuria associated with Srl is unknown. In this study, long-term cardiac transplant patients were switched from cyclosporine to Srl-based IS. Concomitant IS consisted of mycophenolate mofetil ± steroids. Proteinuria increased significantly from a median of 0.13 g/day (range 0–5.7) preswitch to 0.23 g/day (0–9.88) at 24 months postswitch (p = 0.0024). Before the switch, 11.5% of patients had high-grade proteinuria (>1.0 g/day); this increased to 22.9% postswitch (p = 0.006). ACE inhibitor and angiotensin-releasing blocker (ARB) therapy reduced proteinuria development. Patients without proteinuria had increased renal function (median 42.5 vs. 64.1, p = 0.25), whereas patients who developed high-grade proteinuria showed decreased renal function at the end of follow-up (median 39.6 vs. 29.2, p = 0.125). Thus, proteinuria may develop in cardiac transplant patients after switch to Srl, which may have an adverse effect on renal function in these patients. Srl should be used with ACEi/ARB therapy and patients monitored for proteinuria and increased renal dysfunction.
A calcineurin-inhibitor (CNI)-based regimen is the cornerstone of immunosuppressive therapy after cardiac transplantation. CNIs have reduced acute rejection and infection and markedly increased survival of cardiac transplantation patients (1,2). However, the dose- and time-dependent nephrotoxic effects of CNIs can limit long-term survival (3–5), and chronic renal failure is a major cause of morbidity and mortality in long-term cardiac transplant patients. In recent analyses of cardiac transplant patients, the rate of end-stage renal failure (ESRF) 5 years after cardiac transplantation was between 3.9% and 10.9% (4). In addition, cardiac transplant patients on chronic hemodialysis had a much higher risk of death (relative risk 4.55) (4–6). Independent risk factors for developing ESRF were pretransplant renal function, postoperative renal failure, diabetes and age (4).
The target of rapamycin (TOR) inhibitor sirolimus, Srl, and its derivative, everolimus, are macrolide antibiotics with antiproliferative and potent antirejection properties (7,8). There have been many studies of the use of CNI-free therapy based on Srl in renal transplant patients (9–11). In cardiac transplantation patients, Srl has been used in combination with mycophenolate-mofetil (MMF) in long-term patients who were weaned from CNIs due to renal insufficiency (12,13), and the early results have been promising.
However, progressive deterioration of renal function has been observed after switch to Srl in a considerable number of renal transplant patients with slowly deteriorating graft function, most with chronic allograft nephropathy (CAN) (14,15). Several studies addressing conversion in renal transplant patients with some renal dysfunction reported an increase in proteinuria after the switch to Srl, in some cases resulting in nephrotic-range urinary protein excretion (14,16,17).
The aim of this study was to determine whether conversion from CNI-based protocols to Srl-based regimens in long-term cardiac transplant patients is associated with an increase in urinary protein excretion, and whether this effect has an impact on renal function.
Patients and Methods
This study is an analysis of patients included in a prospective controlled switch protocol after cardiac transplantation at the Medical University of Vienna. Written informed consent was obtained from all patients in this study.
Inclusion and exclusion criteria
All patients were >18 years old and more than 12 months after cardiac transplantation. Inclusion criteria included one of the following: CNI-associated chronic renal impairment, defined as serum creatinine >1.7 mg/dL for >6 months; or transplant vasculopathy, defined as ≥50% stenosis in one or more coronary vessels, as detected by routine angiographic examination; or skin tumors detected during yearly routine dermatological examinations.
All patients with renal impairment were on a ‘CNI-sparing protocol’ for at least 6 months before switch. The CNI-sparing protocol consisted of low-dose cyclosporine A (CsA; 50–100 ng/mL) in combination with MMF (1–2 g/day) ± low-dose steroids (0–5 mg/day). Use of other nephrotoxic drugs had been minimized in all patients before enrolment.
The reasons for renal failure other than CNI-induced nephropathy included renal cysts, ureter obstruction and renal artery stenosis, which were characterized using ultrasound, Doppler, laboratory testing and clinical history. These patients were excluded from the switch protocol. Patients with hyperlipidemia requiring more than statin therapy or the presence of an abnormal blood count with significant anemia, leucopenia or thrombocytopenia were also excluded. Furthermore, included patients had no evidence of cellular cardiac rejection requiring treatment in the year before enrolment and no signs of active infection at the time of switch. Patients were included only if proteinuria was measured by 24-h urine collection preswitch and postswitch.
Immunosuppression conversion was conducted over a 4-week period in our outpatient clinic (Figure 1). The first week, patients were given a 3 mg oral dose of Srl (Rapamune®, Wyeth-Ayerst Pharmaceuticals, Collegeville, PA) at least 4 hours after their morning dose of CsA. At the same time, the CsA dosage was reduced by 50%, and the MMF and steroids remained unchanged. After 1 week, Srl trough levels were measured; if Srl levels were in the target range of 5–10 ng/mL, CsA was discontinued. If trough levels were below the target level, Srl was increased by 1 mg and CsA therapy was continued until the next Srl measurement 1 week later. The first 20 patients undergoing the switch protocol underwent an endomyocardial biopsy and echocardiographic examination 2 weeks after cessation of CsA. Over the next 6 months, a cardiac ultrasound assessment was performed on a monthly basis. After 20 patients were studied, routine endomyocardial biopsies were only performed if acute rejection was suspected (after clinical or echocardiographic examination).
All patients received statin therapy with atorvastatin (10–20 mg/day). Fifty-four patients (89%) were already receiving antihypertensive medication prior to switch to Srl. Of these, 58% were treated with ACE-inhibitor (ACEi) (n = 32) and 7% (n = 4) patients were being treated with a combination of ACEi and angiotensin receptor blockers (ARB). Antihypertensive therapy was not changed during follow-up.
Weekly visits were mandatory for the first month after switch, followed by monthly visits for 6 months and visits every 2 months thereafter. Routine measurements included physical examination, blood pressure measurement, chest x-ray and routine laboratory testing. Renal evaluation included 24-h urine volume measurement, urinary sediment, full laboratory analysis, renal ultrasound and Doppler analysis. Twenty-four-hour urine measurements of protein excretion were performed preswitch and 3, 6, 12 and 24 months postswitch.
Continuous variables with normal distribution are expressed as mean ± SD, all others are expressed as median and range (‘minimum–maximum’). Creatinine clearance and proteinuria changes over time were compared by sign test. Differences between groups were tested using the Kruskal-Wallis test. Patients were divided into three groups according to their level of urinary protein excretion just before the conversion. Patients with proteinuria ≤0.2 g/day were assigned to the ‘no/low-grade proteinuria’ group. Patients with proteinuria >0.2 g/day and <1.0 g/day were assigned to the ‘medium-grade proteinuria’ group. Patients with >1.0 g/day proteinuria were assigned to the ‘high-grade proteinuria’ group. Changes from one group to another were tested by McNemar's test. A p-value <0.05 was considered to be statistically significant.
Between March 2002 and November 2005, 80 long-term cardiac transplant recipients were switched to Srl-based immunosuppression. Of these, 61 (55 male, 6 female) were included in the analysis. The main reasons for exclusion were early intolerance to Srl (n = 5), no preswitch 24-h urine measurements (n = 7), no postswitch 24-h urine measurements (n = 3) and other reasons (n = 4).
The switch to Srl was performed 8.1 ± 4.5 years after transplantation (range 0.5–19 years). Patient characteristics are summarized in Table 1. The main reasons for switch were chronic renal insufficiency (n = 49) followed by graft vasculopathy (n = 7) and skin tumors (n = 5). Average CsA levels before switch were 84.3 ± 37.7 ng/mL (range 25–184). All patients received MMF (an average of 1720 ± 2990 mg; range 500–2000 mg) and 75.8% received low-dose steroids (average 3.2 ± 1.4 mg/day; range 0–5 mg/day).
Table 1. Patient characteristics
Data are reported as the mean ± SD, median (min-max) or absolute numbers (percentage), as indicated.
A total of three patients died during 36-month follow-up. Overall actuarial 12-, 24- and 36-month survival was 100%, 98.1% and 95.2.8%, respectively. The cause of death was infection (n = 2) and graft vasculopathy (n = 1).
Three patients became dialysis-dependent during follow-up, and were started on dialysis 3 months (n = 1) or 12 months (n = 2) after switch to Srl. Two of these patients died after starting dialysis due to infection (11 and 27 months after dialysis was started). The other patient received a renal transplant 48 months after the switch (36 months after the start of dialysis).
Two of the 61 patients had to be switched back from Srl to CNI therapy. One patient withdrew consent, and the other patient developed interstitial nephritis 8 months after switch to Srl.
All 20 patients that received routine postswitch endomyocardial biopsies showed no signs of clinical rejection (ISHLT grade G0: n = 18, G1: n = 2 [1a using the old classification]). All other patients showed no signs of acute rejection in serial echocardiographic examinations.
Overall, Srl therapy was well tolerated, and there was a low incidence of the side effects that are typical for Srl. Some patients (25%) had gastrointestinal side effects (e.g. diarrhea and/or nausea) early after switch to Srl. Lymphedema in the arms or legs were observed in 15% of the patients. Acne and aphthous ulcers in the mouth affected 5% of patients. All side effects were easily managed by either Srl or MMF dose adaptation.
The median baseline proteinuria was 0.13 g/day (range: 0–5.7). Proteinuria was significantly different before and after the switch (p = 0.0024) (Figure 2), and we observed that it increased to a median of 0.15 g/day at 3 months (0–4.98, p = 0.3594), to 0.16 g/day at 6 months (0–7.25, p = 0.3594), to 0.16 g/day at 12 months (0–7.25, p = 0.2088) and to 0.23 g/day at 24 months (0–9.88, p = 0.0024). Forty-five patients had an increase in proteinuria after the switch, 6 patients showed no change and 13 patients had a decrease. There was a median decrease in proteinuria of 0.12 (0.01–2.67). Before the switch, patients with decreasing proteinuria had similar renal function as patients with increasing proteinuria (57.2 ± 23.6 vs. 52.7 ± 24.4 mL/min; n.s.). Overall, there was a significant change from lower-grade proteinuria to higher-grade proteinuria over the course of time. Proteinuria pre- and postswitch was as follows: preswitch: 68.8% no/low grade proteinuria, 19.7%, medium grade and 11.5% high grade; 24-month postswitch: 44.3% no/low-grade proteinuria, 32.8% medium grade and 22.9% high grade; p = 0.006 (Figure 3). We noted that some individual patients (n = 4) who had no significant proteinuria before the switch developed high-grade proteinuria (2.65 ± 1.34 g/day) after the switch (Table 2). The average time to development of higher-level proteinuria was 10.7 ± 9.84 months after switch (median: 7 months). There was no difference in the development time of proteinuria from low-grade to medium- or high-grade versus from medium-grade to high-grade proteinuria.
Patients with established transplant vasculoptathy before switch, had a significant increase in proteinuria during follow-up (pre: 0.12 [0–0.19] vs. post 0.19 [0.13–0.35]; p = 0.0209). Yet, none of the patients developed high-grade proteinuria.
Patients who received ACEi and/or ARB's during the entire follow-up period showed no changes in proteinuria, whereas patients not receiving this therapy showed a significant increase in proteinuria. After 24 months, these two groups differed significantly in their proteinuria levels (Table 3).
Table 3. Development of proteinuria with and without ACE-inhibitor/angiotensin II blocker therapy
p-value pre- vs post
Median proteinuria in g/24 h (range); ACEi = Angiotensin converting enzyme inhibitors; ARB = Angiotensin II blocker therapy.
p-value ACEi/ARB vs. no therapy
In the entire study population, creatinine clearance remained unchanged after conversion (Figure 4). Yet there was an early slight increase, which remained stable during the first year after the switch. After the first year, creatinine clearance decreased to almost preswitch levels. Patients who were switched due to skin cancer or graft vasculopathy had significantly better renal function prior to switch than patients who were switched due to renal insufficiency (82.6; 68.5–132.1 vs. 42.4; 17.3–61.5; p < 0.001). After 24 months, there were no significant changes in renal function in either group (76.6; 58.4–184.6 vs. 45.1; 9.0–81.0). Development of proteinuria after switch had a marked impact on the course of renal function (Figure 5). Whereas patients who remained in the no/low-grade proteinuria group after switch showed an increase in renal function over time, there was no benefit for patients who developed medium- to high-grade proteinuria. In patients with low-grade or no proteinuria before the switch, renal function increased immediately after the switch and remained good until the end of observation time (preswitch: median 42.5 mL/min; range: 26.2–120 mL/min) versus 24 months post (64.1 mL/min; range 28.8–185 mL/min, p = 0.25). In contrast, patients who developed medium-grade proteinuria showed an early increase in renal function, followed by a stable but slow decline until month 24. At the end of follow-up, these patients had preswitch renal function. Prior to the switch both patient groups showed no difference in creatinine-clearance. In contrast, patients who later on developed high-grade proteinuria had worse renal function before the switch (39.6 mL/min, range 17.3–52.4 mL/min) compared to the two other groups (no/low proteinuria group: 42.5 mL/min; range: 26.2–120 mL/min; medium proteinuria group: 45.2 mL/min; range: 24.1–78.3 mL/min; p = 0.09). Patients with high-grade proteinuria had a slight increase in renal function early after the switch and remained stable for the rest of the first year. However, after 24 months these patients had decreased renal function (29.2 mL/min; range: 6.6–55 mL/min; p = 0.125); in fact, renal function was even worse than before switch. During the first year after switch, renal function was comparable between the three groups. In contrast, beginning at 12 months postswitch, renal function was significantly worse in the high-grade proteinuria group compared to the two other groups after 24 months (p < 0.0001; Figure 5).
This is the first study of proteinuria in long-term cardiac transplant patients after conversion from CNI-based to Srl-based immunosuppression. Patients who developed high-grade proteinuria after switch had a significant decrease in renal function. In contrast, patients who remained without proteinuria had significantly better creatinine clearance at the end of follow-up.
Prospective studies have shown that converting cardiac transplant patients from CNIs to Srl and MMF results in improved renal function without compromising allograft function (12,13,18). Yet two of the larger studies showed patient sub-populations that did not benefit from switch to Srl: both Groetzner and Kushwaha noted that 10–15% of the patients had a decline in creatinine clearance after the switch (12,13). Both authors considered the most likely explanation to be irreversible chronic renal damage due to CNI toxicity, but renal protein excretion was not measured in these studies.
Proteinuria has emerged as a problem for renal transplant patients converted to Srl (14,16,19). Proteinuria after cardiac transplantation has been reported, mainly as a sign of renal dysfunction (20,21). Although 31% of the patients in this study had proteinuria before conversion, we observed an increase of proteinuria in approximately two-thirds of the patients after switch to Srl. In 78% of the patients with increased proteinuria, the increase was >100%. Remarkably, even the 43% of patients with no/low proteinuria before switch experienced an increase of more than 300 mg/day in urinary protein excretion after conversion. These findings are consistent with results from previous studies that showed a significant increase in urine secretion in renal transplant patients after switch to Srl (14,19,22). Nevertheless, only 4 patients (6.5%) without preswitch proteinuria developed high-grade proteinuria. Importantly, the occurrence of proteinuria seemed to be directly related to a decrease in renal function, i.e. renal function deteriorated after proteinuria onset. Most patients had a marked early benefit after conversion, even though they later developed higher-grade proteinuria in combination with worsening renal function. Therefore, the advent of higher-grade proteinuria seems to be associated with decreased renal function in Srl-treated patients.
In renal transplantation, proteinuria can be a sign of CAN (23). In contrast, after cardiac transplantation, increased urinary protein excretion occurs in the native kidney and must therefore be a sign of non-allograft-associated pathology. Thus proteinuria might be a sign that preexisting renal damage is progressing, or it could be a sign of renal injury caused by Srl-associated toxicity. The long-term advantage of Srl-based immunosuppression may therefore be offset by the higher prevalence of proteinuria. Renal transplantation studies report an incidence of newly developed proteinuria between 25% and 50% (14,19,22,24–26). These reports did not determine whether proteinuria was caused by Srl itself or whether it was the result of CNI withdrawal. Switch from CNIs might unmask existing altered glomerular permeability to serum proteins by increased renal blood flow and glomerular pressure. One study found that conversion of cardiac transplant recipients from CNI- to azathioprine-based treatment for chronic kidney dysfunction also led to an increase of proteinuria (27). Hemodynamic changes in the glomerulus are suggested as a possible explanation. However, another study of conversion from CNI-based to CNI-free MMF-based treatment for CAN showed a decrease of proteinuria after conversion (28). Moreover, van den Akker et al. described an increase of proteinuria in patients converted to Srl from an azathioprine-based protocol (26). Our data also suggest another cause for proteinuria, as there was an average of 7 months between switch to Srl and onset of proteinuria: thus, a change in intrarenal hemodynamics might not be sufficient to explain proteinuria in these patients. Conversely, proteinuria may have resulted from an Srl-specific effect leading to glomerulopathy or tubulointerstitial disease (16,29,30). Dittrich et al., as well as Izzedine et al., identified various glomerular diseases as responsible for an increase of proteinuria in patients switched to Srl (16,31). In addition, Diekmann and colleagues suggested that Srl might contribute indirectly to a mechanism leading to genuine glomerular damage (32). However, there is little experimental data that supports the hypothesis that Srl is directly toxic to glomeruli (33,34). In contrast, Straathof-Galema described tubular mechanisms as possible explanation for proteinuria (29). Potential mechanisms include decreased tubular reabsorption or altered protein transportation (30). Other possible explanations have been suggested based on experimental data. In vitro studies by Lieberthal showed that Srl induces apoptosis and cell-cycle arrest in mouse proximal tubular cells (35). On the other hand, studies by Coombes et al. have demonstrated that rapamycin induces renal injury by intratubular obstruction, secondary to increased protein cast formation (36). Our study cannot answer these complex questions, as it was not designed to elucidate the pathophysiological mechanisms of Srl-associated proteinuria. Another limitation of this study was the absence of routine renal biopsies, which would have provided more insights into pathophysiological changes accompanying or resulting from the development of proteinuria. Therefore, the mechanism(s) of proteinuria development in native kidneys caused by Srl therapy remain speculative.
The rate of proteinuria is reported to be lower if ACEi or ARB's are used in SRL-treated renal transplant patients (37). This is in agreement with earlier reports of the antiproteinuric effect of ACEi/ARB therapy in diabetic- and non-diabetic nephropathy (38–40). Due to the high incidence of hypertension after cardiac transplantation, most patients receive antihypertensive medication (2). In this study, 68% of patients were treated with either ACEi therapy, or a combination of ACEi and ARB therapy, before switch and during the entire follow-up. Similar to renal transplant, proteinuria did not change significantly in the ACEi/ARB group, whereas there was a significant increase of proteinuria in patients without ACEi/ARB therapy. Saurina et al. have shown that intraglomerular pressure increases and renal functional reserve decreases after switch to Srl. Both these parameters are typical signs of hyperfiltration, and therefore use of ACEi and/or ARBs might be feasible treatment options (24). However, more experience and longer follow-up is needed to examine whether there is a nephroprotective effect of ACEi/ARB therapy in combination with Srl after cardiac transplantation. Another recent analysis showed that statin therapy was associated with markedly lower incidence of proteinuria in renal transplant patients using Srl therapy (41). There is clear evidence that statin therapy reduces the risk of allograft vasculopathy and improves long-term survival after cardiac transplantation (42). Therefore, almost all cardiac transplant patients are treated with statins as prophylactic therapy. As all of our patients received statins, we can only speculate whether development of proteinuria would have been different without statin use.
To make matters even more complex, there no consistent pattern emerged in how patients reacted to the switch to Srl. Thirty-five percent of our patients did not show an increase in proteinuria; of these, 57% actually showed a reduction of proteinuria after the switch to Srl. Nevertheless, only two patients changed into a lower proteinuria group (both medium to low group). Therefore, different groups of patients seem to react differently, to a switch to Srl. However, we could not identify the factors associated with either an increase or a decrease in proteinuria after conversion to Srl. Until there is greater understanding of the pathophysiology underlying proteinuria in Srl-treated patients, conversion protocols should be performed with caution. We urge physicians to take these precautions to minimize potential problems after conversion to Srl: first, measure proteinuria preswitch; second, perform serial postswitch assessments of proteinuria; and third, if high-grade proteinuria develops, closely monitor renal function. Although 24-h urine measurements have been used in this study, this might be too complicated for general use in clinical settings; 24-h urine collections are inconvenient for many patients and are sometimes inaccurate due to improper collection. In most cases, screening with urine dipsticks is acceptable for detecting proteinuria (43). Quantification of proteinuria can be done by spot morning protein/creatinine ratio, which is closely associated with protein excretion measured in a 24-h sample (43,44). Initiation of ACEi and/or ARB therapy might be beneficial in some patients, although there is not enough data regarding this yet for cardiac transplantation patients. Finally, if renal function decreases in patients who have developed proteinuria, reconversion to a CNI-based regimen should be considered.
In summary, our study found that long-term cardiac transplant patients who are switched from CNI-based to Srl-based immunosuppression showed a significant increase in proteinuria. Patients who developed high-grade proteinuria (>1 g/day) had a significant decrease in renal function, whereas those who did not develop proteinuria had a significant benefit from Srl therapy.