Islet cell transplantation (ICT) has emerged over the past decade as a treatment option for type 1 diabetes. Most centers have focused on achieving insulin independence and reducing glycemic lability and severe hypoglycemia (1). The effect of ICT on chronic diabetic complications has not been as well studied.
Although intensive medical therapy has improved the prognosis, diabetes remains the leading cause of end-stage renal disease (2), nontraumatic lower limb amputations (3), and visual loss in working-age adults (4). Intensive medical treatment increases the risk of severe hypoglycemia (5) and good glycemic control is difficult to maintain over long periods (6).
We have been examining the effects of medical therapy and ICT on the progression of microvascular complications and present our latest results in this report.
Patient Characteristics and Follow-Up
At entry, the 45 subjects (21 men) were 46.6±8.5 years old with a diabetes duration of 29.8±9.2 years and body mass index 25.8±3.5 kg/m2. The median ACR was 8.0 mg/mmol (IQR 3.1–25.4) with overt proteinuria (ACR >20 mg/mmol) present in 12 (27%). The average insulin dose at the time of first ICT was 42.6±14.3 units/day and was 46.5±16.8 units/day for the medical subjects at their last visit. The patient flow is outlined in Table 1. The first ICT was performed in March 2003 and as of June 2010, 32 subjects have received 85 islet infusions: 3 have received 1, 12 received 2, 10 received 3, and 7 received 4. Subjects have received (mean±standard deviation) 1,276,606±401,316 IE in total. Nine no longer receive immunosuppression: two had primary graft failure after their first infusion and one of these has withdrawn from the study, two are lost to follow-up, two stopped because of persistent fatigue, two developed malignancy (one skin and one breast), and one had a severe cytomegalovirus infection.
The median follow-up as of July 2010 is 47 months for medical patients and 66 months post-ICT. The discrepancy in follow-up duration is due to the one-way crossover design and increases with time. For transplanted subjects, the median time from study entry to first transplant was 21 months. Currently, 8 medical and 29 transplanted subjects are being followed with the remaining 8 (5 medical, 3 post-ICT) lost to follow-up.
Graft Function and Metabolic Status
All of the 23 subjects who were maintained on immunosuppression had persistently detectable C-peptide secretion. Twenty-two have stopped insulin for more than or equal to 3 months and 12 are currently not requiring insulin. The longest a subject has been insulin independent is 86 months.
HbA1c at study entry was 8.1%±1.2% and this improved to 7.0%±0.7% at the time of first ICT. The overall average A1c post-ICT was 6.7%±0.2% and 7.8%±0.3% in the medical patients (P<0.001).
Systolic blood pressure was significantly lower post-ICT group than with medical treatment (122±7 mm Hg vs. 130±10 mm Hg, P<0.001) although diastolic values were similar (70±4 mm Hg vs. 73±5 mm Hg).
Table 2 shows that the rate of decline in GFR is more rapid for medically treated subjects than post-ICT. This finding applies whether GFR is measured by 99mTc-DTPA or estimated by the modification of diet in renal disease equation. There is no difference in the results when only subjects with more than or equal to 2 or 3 years of follow-up are included. The rate of decline in the 13 medical subjects who have not been transplanted (−2.4 mL/min/1.73 m2/year) is not different from the entire group. Sixteen subjects had sufficient GFR measurements in both the medical and post-ICT phases to allow an intraindividual comparison using a paired t test. The median (interquartile range) decline during medical treatment was −6.7 mL/min/1.73 m2/year (−2.5 to −12.2 mL/min/1.73 m2/year) and −1.3 mL/min/1.73 m2/year (−4.1 to 0.1 mL/min/1.73 m2/year) post-ICT (P=0.01). The median ACR is 2.0 mg/mmol in each group at the time of last measurement. Of the 12 subjects with proteinuria at study entry, 3 of 4 medical and 7 of 8 post-ICT have regressed to microalbuminuria. The degree of proteinuria at study entry did not affect the rate of decline in GFR (data not shown).
Table 3 shows that retinopathy progression is significantly more likely during medical therapy than after ICT (P<0.01). There has been no progression in eyes with mild NPDR. One medically treated subject progressed from moderate NPDR and two from severe NPDR to PDR and were treated with laser. Seven eyes that had PDR at study entry also required laser. Laser was given for accepted indications (7) and was initially performed from 4 to 40 months after entering medical therapy. Since our previous report, additional laser has been given to four eyes in the medical group, all having had previous laser during the study. As reported before, two eyes in the medical group received laser for clinically significant macular edema.
Figure 1 shows fairly stable NCV over time in both groups. There is a trend favoring the post-ICT group that does not reach statistical significance (P=0.07). The degree of neuropathy at study entry did not affect response to therapy (data not shown).
The results of our prospective, crossover study demonstrate that there is less progression of diabetic retinopathy and nephropathy after ICT than with intensive medical therapy. We chose a one-way crossover design (8) for the study because it allows for both a small sample size and patients to serve as their own controls, important features when ICT has limited availability and there is wide individual variability in the rate of progression of microvascular complications (9). To be valid, this design requires that there is no carryover effect of the first treatment on the second. We believe that this is a valid assumption for microvascular complications, for example, supported by the demonstration that the rate of change in GFR does not vary over time (10).
This report extends our previous finding (11) that ICT does not have a deleterious effect on the renal function of native kidneys in type 1 diabetes. In fact, we have demonstrated that ICT slows the rate of decline in GFR compared with intensive medical therapy in patients with microalbuminuria or overt proteinuria at study entry. The finding is similar whether the subjects are analyzed as a group or as a within patient comparison of medical and post-ICT function. The expected (9) variability in the rate of decline between patients is shown by the wide confidence intervals (Table 2) and interquartile range. Most reports of renal function after ICT have suggested worsened renal function, at times progressing to end-stage renal disease (12–15). We believe that the rate of decline in the medical group is what would be expected from the literature for patients with type 1 diabetes and this degree of proteinuria (2, 9, 10, 16).
We suggest that our choice of maintenance immunosuppression may be an explanation for our improved results. Both tacrolimus (17) and sirolimus (18) are individually nephrotoxic and sirolimus may potentiate the nephrotoxicity of tacrolimus (19). The combination of tacrolimus-MMF has been shown to be less nephrotoxic than tacrolimus-sirolimus for both native (20) and transplanted (21, 22) kidneys. Previous studies (12–15) had more exposure to a calcineurin inhibitor + sirolimus/everolimus than our subjects, although many converted some subjects to MMF. Senior et al. (23) reported reversal of proteinuria in ICT recipients when sirolimus was replaced with MMF providing direct evidence of the toxicity of tacrolimus-sirolimus in an ICT population.
Our retinopathy results have been published previously (24). Since that time, there has been additional progression in subjects treated medically but no evidence of progression post-ICT after a median follow-up of 67 months. As expected, most progression occurred in the more advanced stages of retinopathy (25), and the rate of progression was similar to other reports (26, 27). Stabilization of retinopathy post-ICT is consistent with that reported with pancreas transplantation (28, 29). One limitation is that determination of retinopathy endpoints was not masked. However, all treatments were given for approved indications, and we did not identify any instance where treatment was held inappropriately. The scale (30) that we used in our small study is not as sensitive as the Early Treatment Diabetic Retinopathy Study (ETDRS) grading system (31), and we would recommend that the gold-standard ETDRS protocols be used in future randomized studies about the effect of ICT on the progression of retinopathy.
Our NCV results showed stability in all subjects, with a nonsignificant trend favoring the transplanted group. Previous studies have reported some neurological improvements after ICT but have not demonstrated an overall significant benefit (32, 33). Because the pancreas transplant literature shows continued improvement over time (34), it is possible that a longer period of observation will yield different results in our patients.
Possible explanations for the good microvascular outcomes in the ICT group include better glucose control (25, 35) or the presence of C-peptide (36, 37). We do not think that the lower systolic blood pressure is a cause given the recent findings from Action to Control Cardiovascular Risk in diabetes (ACCORD) (38).
The best use of ICT in the treatment of type 1 diabetes continues to be debated (39). A major question is whether ICT can slow the progression of microvascular complications because current medical therapy is only partially effective. For example, there has been no reduction in the need for renal replacement therapy for type 1 diabetes and those requiring dialysis have a 5-year mortality of 76% (40). Our current study suggests that ICT can improve this prognosis. However, it must be considered to be hypothesis generating because of its crossover design and small enrollment. We believe that there need to be multicenter, randomized, controlled trials to answer this question in a definitive way.
MATERIALS AND METHODS
We conducted a prospective, one-way crossover, cohort study comparing intensive medical therapy and ICT on the progression of complications in patients with type 1 diabetes.
Eligible subjects were 20 to 65 years of age, were C-peptide negative, and had evidence of any grade of retinopathy plus mild nephropathy (urine albumin/creatinine ratio [ACR] >2.0 mg/mmol and glomerular filtration rate [GFR] >70 mL/min). Forty-five subjects enrolled between January 2002 and January 2005 and are the subject of this report.
All subjects began with a 3-month run-in phase to ensure suitability for our program. This included beginning intensive medical therapy (consisting of intensive glucose management, angiotensin blockade, and control of blood pressure and lipids to recommended levels) and ensuring suitability for an islet infusion. Data collection began following study enrollment. Insulin adjustment followed established methods (41, 42) with individual glucose targets balancing the goals of optimal glycosylated hemoglobin (HbA1c) levels and minimal hypoglycemia. Patients had phone contact with diabetes nurse educators every 2 weeks and clinic visits every 3 months.
Upon receipt of a donor, the best-matched subject received the ICT. Islet isolation and infusion were performed as described (43). Each subject was offered up to three islet infusions and a total of more than 12,000 islet equivalents (IE)/kg to try to achieve insulin independence. If subjects became insulin independent, and then over time needed to restart insulin, they were offered additional infusions in an attempt to regain insulin independence.
Immunosuppression was induced with antithymocyte globulin for the first transplant (1 mg/kg/day for 5 days) and basiliximab (20 mg on days 1 and 4) for subsequent infusions. All received tacrolimus as part of their maintenance immunosuppression. Subjects received 2 to 12 mg of tacrolimus/day targeting trough levels 8 to 10 ng/mL for the first month, 6 to 8 ng/mL until 90 days, and then 4 to 6 ng/mL. Initially sirolimus (2–3 mg/day targeting trough levels 10–12 ng/mL for the first month and then 5–8 ng/mL) was administered with tacrolimus, but we found that this combination was poorly tolerated by most subjects. Mycophenolate mofetil (MMF; 1–2 g/day) was substituted and is now routinely used with tacrolimus. Of the subjects reported in this manuscript, two subjects received tacrolimus and sirolimus since their initial transplant because of excellent results (off insulin for 88 and 44 months) and one is on sirolimus and MMF because tacrolimus was suspected of causing hallucinations. Three subjects received tacrolimus and sirolimus for a maximum of 8, 13, and 35 months before switching, while the other 26 have only received tacrolimus and MMF.
The predefined primary renal endpoint was the rate of change in GFR as measured by the blood clearance of 99mTc-diethylenetriaminepentaacetate (DTPA), every 6 months in the medical and post-ICT groups (44, 45). Direct measurement of GFR is superior to estimates based on serum creatinine in clinical trials (46), especially in patients with normal GFR (47) and those with diabetes (48). Secondary endpoints were the rate of change in estimated GFR (49) and urinary albumin excretion measured by ACR (normal <2.0 mg/mmol).
The rate of change of GFR was determined by the slope of the regression line in each group. The analysis of slope is believed to be better than a threshold approach when assessing the effect of risk factors on renal function (50) and is particularly important when there is a variable length of follow-up between groups. To be included in the analysis, a subject had to have at least three measurements of GFR over at least 12 months in a treatment group (30 subjects in the medical group and 29 post-ICT met this criteria). Review of recent studies suggests an average rate of decline in GFR of 1 mL/min/year in the general population, 1 to 2 mL/min/year in patients with microalbuminuria, and 4 to 5 mL/min/year with overt proteinuria (ACR >20, equivalent to albumin excretion >300 mg/day) (2, 9, 16, 51). With 27% of our cohort having overt proteinuria, we estimated a rate of decline in GFR for the group of approximately 2.5 mL/min/year. We also compared the rate of decline during medical and post-ICT periods for the subjects who had sufficient measurements in each group.
Ophthamologic assessment was performed by a retina specialist who was aware of their treatment category. Annual seven-field stereo fundus photographs were performed by a certified photographer (31). Severity of retinopathy was assessed using the International Scale which has three levels of nonproliferative diabetic retinopathy (NPDR) (mild, moderate, and severe) and one level of proliferative diabetic retinopathy (PDR) (30). Macular edema was classified as clinically significant or not (52). Grading was performed to compare levels of severity at entry into medical therapy, just before initial ICT and at the last visit. Predefined endpoints for each eye were (a) NPDR advancing by more than or equal to 1 level of severity, (b) progression of PDR to a severity level that qualified for scatter laser, or (c) development of macular edema with visual acuity less than or equal to 20/40, which qualified for focal or grid laser treatment.
Nerve conduction studies are the most reliable method to study diabetic polyneuropathy in clinical trials (53, 54). While a number of parameters are assessed during the test, nerve conduction velocity (NCV) is the most reproducible and widely used measurement and is used in this report (55). Testing of seven nerves (sensory: ulnar, median, and sural; motor: ulnar, median, peroneal, and tibial) every 12 months was performed in the same laboratory under standardized conditions. The reporting neurologist was not blinded as to whether a patient had received an ICT.
The primary endpoint was the rate of change in NCV in the medical and post-ICT groups. The NCV from each nerve for all subjects in the group were averaged to produce a single value for each year. Nerves for which no signal could be obtained were excluded from analysis. The slopes were calculated by simple linear regression to produce an annual rate of change.
Analysis was by intention-to-treat. Patients crossed over and began to be analyzed in the ICT group once they received their first islet infusion. Data for continuous variables with a normal distribution are presented as mean±standard deviation and otherwise as median and interquartile range. Continuous variables with a normal distribution were compared using a two-tailed, paired t test, and those not normally distributed using the Mann-Whitney U test. Categorical variables are compared using chi-square test.
All subjects gave written informed consent and the study was approved by the institutional review board of the University of British Columbia.
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