This analysis was performed using data from the ongoing National Institutes of Health sponsored Angiotensin II Blockade for Chronic Allograft Nephropathy Trial (clinicaltrials.gov NCT 00067990). In this 5-year long trial, 153 kidney transplant recipients were randomized to placebo or losartan at 58 ± 34 days after receiving a living- or a deceased-donor transplant. Recipients were transplanted at either the University of Minnesota (UMN) or Hennepin County Medical Center (HCMC), Minneapolis, MN. The primary endpoint of this trial was the change in cortical interstitial volume from postperfusion allograft biopsies to those performed at 5 years after transplantation. Exclusion criteria included: age < 18 years, serum creatinine ≥ 2.5 mg/dL, potassium > 5.4 mEq/L, known hypersensitivity to losartan or iodine allergy, documented renal artery stenosis, recipients of grafts from an HLA-identical sibling, recipients whose primary renal disease is primary hyperoxaluria, dense-deposit disease, focal segmental glomerulosclerosis or hemolytic uremic syndrome, women of childbearing age who wished to become pregnant and/or were unwilling to use contraceptive measures or who were pregnant and had a cardiovascular indication for ACE inhibitors or AII blockers. Those transplanted at the UMN received induction with five doses of Thymoglobulin® (Genzyme) and a 5-day course of steroids in tapering doses (methylprednisolone 500 mg, given in the operating room, followed by prednisone, 1 mg/kg on postoperative day 1; 0.5 mg/kg on days 2 and 3; and 0.25 mg/kg on days 4 and 5). Recipients who were on prednisone prior the transplantation (mainly for lupus or having a prior functioning allograft) remained on previous maintenance dose. For maintenance therapy, patients received a combination of a calcineurin inhibitor (cyclosporine or tacrolimus) with mycophenolate mofetil or sirolimus. Rejection episodes were treated with a rapid steroid taper (methylprednisolone 500 mg × 3 days followed by prednisone taper over 10–14 days down to 5 mg daily indefinitely) or Thymoglobulin® for 7–10 days for steroid-resistant or histologically moderate-severe rejection episodes and remained on maintenance steroids as well. Recipients transplanted at HCMC, in contrast, received induction therapy with two doses of an interleukin 2 antagonist: Basiliximab® (Novartis) or Daclizumab® (Roche), and Solumedrol 500 mg IV × 3 doses followed by prednisone taper down to maintenance dose 12 mg by 6 months. For maintenance therapy, they received a calcineurin inhibitor, mycophenolate mofetil and prednisone. Rejection episodes were treated with high dose of steroids or Thymoglobulin based on severity.
The steroid group (Group 1) consists of UMN recipients who received steroids for 5 days only in perioperative period, and therefore those who received additional steroids beyond that time either for treatment of rejection or were on maintenance prednisone therapy for other reasons were excluded from this analysis (Figure ). In contrast, recipients from the HCMC program, who remained on maintenance steroids per their protocol, constitute our steroid maintenance group (Group 2). The steroid-free group (n = 107) were the main interest of this analysis but were also compared to 27 contemporaneous renal transplant recipients from Hennepin County Medical Center who were maintained on steroids. To guard against the possibility that these recipients may not have reached a steady state and the exposure to steroids may have been too brief at the time of the first iGFR to cause a significant muscle wasting, we studied 81 of the University of Minnesota recipients who remained steroid free at 1 year (26 recipients either missed their 1-year GFR or dropped out of the study) after the first measurement and 16 HCMC recipients (11 recipients missed their 1-year GFR) who remained on steroids between the two measurements (Figure ). All recipients were on single-strength trimethoprim-sulfamethoxazole, and none were on cimetidine.
All trial participants underwent direct measurement of GFR by iothalamate clearance (iGFR) 58 ± 34 days posttransplantation at which time they were also randomized to losartan or placebo. A second iGFR was performed 11–13 months after the one performed at randomization. Iothalamate GFR was done after giving an intravenous loading dose of iothalamate followed by maintenance infusion. All recipients received a water load of 10 ml/kg to maintain urine flow rates at >3 ml/kg/h. If this flow rate was not achieved, an additional water load of 5 ml/kg was given. Five timed urine and plasma collections were performed, and the average of these was calculated. The CV of iGFR was <10%, and GFR was corrected to body surface area (BSA) of 1.73 m2.
Serum creatinine was obtained on the morning of the iGFR, after an 8–12-h fast, and was calibrated against the Cleveland Clinic Biochemistry Laboratory (Cleveland, OH) where serum creatinine was assayed for the Modification of Diet in Renal Disease (MDRD) study as previously described [6–8
]. In May 2008, the creatinine assay at our institution changed from the Jaffe’/CXR Synchron method to the isotope-dilution mass spectroscopy (IDMS)-traceable creatinine [9,10
]. The laboratory provided us with a formula to convert the Jaffe’ assay-based creatinine to the IDMS-traceable creatinine. We used this formula to convert all creatinine values to IDMS-traceable values. To verify the accuracy of the conversion formula in these recipients, we randomly selected 30 serum samples from the pool of 153 recipients and measured serum creatinine using the new IDMS-traceable method, and they were identical. Since values provided by the regression formula provided serum creatinine values that were identical to the directly measured ones, we used the values obtained from it in the 153 recipients.
GFR was estimated using the re-expressed Cockcroft–Gault equation for estimation of GFR for use with standardized creatinine (eGFRCG), the Mayo Clinic equation (eGFRMC) and the MDRD study equation (eGFRMDRD). eGFRCG was calculated using the formula (140 − age) × weight/(72 × SCr) × (0.85 if female) (
] multiplied by 0.8 to correct for the bias in the MDRD study sample [9
]. It is of note that only standardized creatinine can be used for this model. eGFRMC was calculated using the quadratic equation that estimates logarithmic GFR from serum creatinine, age and gender and after indirectly calibrating serum creatinine by applying the following regression relation: IDMS-traceable creatinine = 0.906[−0.213 + (1.098 × Mayo Clinic creatinine)] [12,13
]. eGFRMDRD was calculated using the formula: GFR = 175 × standardized SCr−1.154 × age−0.203 × 1.212 (if black) × 0.742 (if female) [14
]. eGFRCKD-EPI was calculated using the formula: 141 × min(SCr/κ, 1)α × max(Scr/κ, 1)−1.209 × 0.993age × 1.018 [if female] × 1.159 [if black], where Scr is serum creatinine, κ is 0.7 for females, 0.9 for males, α is −0.329 for females and −0.411 for males, min indicates the minimum of Scr/κ or 1 and max indicates the maximum of Scr/κ or 1 [15
We assessed the performance of the four models against iGFR early after transplant and a year later by formally testing the differences in bias, precision and relative accuracy calculated as follows: bias, the average prediction error = Σ (eGFR − iGFR)/n, where n is the number of GFR studies performed and iGFR is iothalamate GFR; relative bias, percent deviation from the iGFR; precision, the value of R2 from the linear regression of iGFR on eGFR; relative accuracy, the percent of estimates falling within 10%, 30% and 50% of iGFR. We also assessed the ability of these four GFR prediction equations to correctly identify recipients with iGFR < 45 and 60 ml/min/1.73 m2
. This was achieved by comparing the proportion of eGFR < 45 and 60 ml/min/1.73 m2
to the proportion of iGFR< 45 and 60, by calculating kappa statistic for agreement and by calculating the sensitivity and specificity of each equation to accurately classify recipients. The equations were compared using a paired t-test for bias, a paired test of proportions for relative accuracy and the Hotelling–Williams test for precision [16–18
]. Statistical significance was assessed with a Bonferroni-adjusted threshold (P
< 0.008) since multiple comparisons were performed. The four equations were ranked from 1 to 4 based on the results of the paired tests, with ‘ties’ assigned the same ranking. Though not formally tested, the interquartile range and the root mean squared error of the bias were computed.
Analyses and graphs were completed using statistical software SAS, version 9.1 (SAS, Cary NC), and R version 2.8.0 (R, Boston, MA).