After the first year of transplantation, chronic allograft nephropathy (CAN) (previously described as “chronic rejection”) is the most important cause of renal graft failure (1–5). Although graft survival is more than 90% for the first year, this is not matched with similar positive outcomes long term. More than 50% of renal grafts fail after 10 years, comprising some 5% of transplant recipients returning to dialysis annually. Angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers reduce proteinuria and delay progression to end-stage renal failure (ESRF) in diseased native kidneys (6–9). These agents, in addition to effectively controlling hypertension and reducing proteinuria, may also block nonpressor effects of angiotensin II on renal cells and particularly the proximal tubules inducing oxidative stress (hypercatabolism), activation of proinflammatory chemokines, cytokines, and transforming growth factor (TGF)-β1 promoting tubulo-interstitial fibrosis (TIF).
Randomized controlled trials (RCTs) using ACE-inhibitors or angiotensin receptor blockers in renal transplant recipients to address the effects on graft function remain relatively limited (systematic review [10]). Data in proteinuric patients with CAN are sparser still (11–15) with no previous RCT. In a series of pilot clinical studies in proteinuric patients, including CAN (13, 16–19), we demonstrated renal proximal tubular peptide hypercatabolism linked with proteinuria, increased urinary ammonia excretion, and markers of tubular injury. These potentially harmful effects were reduced using an ACE-inhibitor (or sodium bicarbonate supplementation [18]).
The main aim of this novel RCT (open design) was to provide evidence-based information on whether lisinopril, an ACE-inhibitor, might ameliorate the rate of decline of renal function in transplant recipients with proteinuria, severe renal impairment, and CAN. The other aims were (1) to provide novel kinetic data on polypeptide catabolism (of radiolabeled aprotinin) before and after 1 year's treatment with lisinopril; (2) to determine the effects of lisinopril on urinary pH, ammonia excretion, and markers of tubular injury, inflammation, and fibrosis; and (3) to perform genotyping studies correlating results with rates of renal graft progression and proteinuria, because polymorphic genes in the ACE pathway might influence response to ACE-inhibitor therapy (20–23). We also uniquely address metabolic acidosis because it is prevalent in transplant recipients (in >60%) (24) and metabolic acidosis increases with ACE-inhibitors (13).
The study design complied with Medical Research Council/Consolidated Standards of Reported Trials guidelines (25) and new UK Medicines and Healthcare products Regulatory Agency regulations 2004 with Clinical Trial Authorisation, and registered 2005 (http://www.controlled-trials.com/ISRCTN76140647). A comprehensive search of the Cochrane library (Databases of systematic reviews and Controlled Trial Register-Renal Group) was undertaken, and the same was also searched from Medline (1990–2008) using key words—renal transplant graft function, progression, survival, ACE-inhibitors, angiotensin receptor blockers, proteinuria, proximal tubule, and catabolism. International/European/National guidelines on best practice were searched and bibliographies from major textbooks studies scanned.
The primary outcome in this study is renal graft progression (mL/min/year). Secondary outcomes are proteinuria, aprotinin catabolic studies, and ACE genotyping. Inclusion criteria are biopsy-proven CAN at least 6 months posttransplantation (deceased donors or live related); not on ACE-inhibitors or angiotensin II antagonists; any combination of immunosuppressive therapy, but not if converted to tacrolimus or mycophenolate mofetil within previous 6 months; proteinuria more than or equal to 1.0 g/24 hr; creatinine clearance more than or equal to 20 mL/min; no history of transient ischemic or cardiovascular event or malignancy in the last 6 months; patients aged between 18 and 70 years (amended 2006 to include >70 years). Exclusion criteria are clinical or histologic evidence of acute graft rejection, renal artery stenosis, persistently high calcineurin-inhibitor levels, abnormal liver function tests, pregnancy, and chronic intractable cough.
Consent
All patients gave informed written consent. There was approval of both the Local Liverpool Research Ethics Committee and of the Administration of Radioactive Substances Advisory Committee.
Sample Size
Based on our pilot data comparing graft progression rates in patients ±ACE-inhibitors (11), the sample size needed was 34 patients, taking the effect size as 10 mL/min/year, SD 8.6, type 1 error=0.05, type 2 error=0.10, and power=90%. We targeted for 40 to 50 patients to allow for dropouts. The stratification randomization method and the allocation sequence were generated independently by the Pharmacy Department. A trial randomization table was used (random permuted blocks), which allowed randomization of patients into blocks of four, two destined to receive lisinopril (group A) and two did not receive lisinopril (group B). The relevant randomization data were recorded and hidden in sealed opaque envelopes and kept in a locked drawer in the Research Office on the Transplant Unit.
Clinical Protocol: Patients
RCT (October 2000–July 2007) was “open” design because ACE-inhibitors have obvious effects on blood pressure and proteinuria. Patients recruited from Merseyside and North Wales were randomized after 5 weeks (run-in period) to group A (given lisinopril) or controls group B and followed up for 1 year. Plasma and 24-hr urine collections for biochemistry, creatinine clearance (CrCl), proteinuria, markers of injury, and pH/ammonia excretion (fresh urine) were performed. Plasma renin and aldosterone levels were also measured. Isotopic 51CrEDTA measurements of glomerular filtration rate (GFR) were made at end of the run-in period, between 4 and 6 months and at 12 months. In group A patients only, radiolabeled aprotinin catabolic studies were performed at similar time points to GFR measurements (see Appendix, Supplemental Digital Content 1,https://links.lww.com/TP/A122).
Lisinopril was commenced at 2.5 mg daily and increased gradually targeting a maximal decrease in proteinuria without symptomatic postural hypotension (average dose 10 mg, 5–27.5, 0.07–0.35 mg/kg/day). We previously showed (13) that lisinopril treatment in patients with CAN led to increased metabolic acidosis (and unacceptable hyperkalemia), which would have precluded safe use. All patients were given oral sodium bicarbonate (titrated to correct metabolic acidosis) and instructed to reduce dietary sodium intake to approximately less than or equal to 80 mmol/day, so that 24-hr urinary sodium excretion would be kept within the reference range, targeting for values of less than 200 mmol/24 hr. Patients with potassium levels more than 5 mmol/L were instructed to reduce potassium intake to 1.0 mmol/kg ideal body weight/day. Patients were on one-alphacalcidol (or calcitriol therapy) if the parathyroid hormone (PTH) level was raised more than 10 pmol/L and given erythropoietin therapy (Aranesp weekly subcutaneous injections) for hemoglobin levels lower than 10 g/dL.
METHODS
Graft Function
Isotopic GFR (51CrEDTA clearance, three sample technique) (3MBq) and index of effective plasma renal flow (tubular function) (99mTc mercapto acetyl tri-glycine two sample technique) (5MBq) were measured as previously (13). Estimated GFR were calculated using two methods (26, 27).
Genotyping DNA Extraction
Genomic DNA was extracted from whole blood using the AutoQ-Chemagic magnetic bead technology with the automated Chemagic Magnetic Separation Module I (www.chemagen.com). A high yield and DNA quality (optical density 260/280>1.8) was achieved with each sample.
SNP Selection and Genotyping
Candidate gene and single-nucleotide polymorphism (SNP) selection was based on literature reports and publicly available databases; SNPdb (www.ncbi.nlm.nih.gov/SNP), HapMap project (www.hapmap.org), OMIM (www.ncbi.nlm.nih.gov/sites/entrez?db=OMIM), and KEGG pathway (www.genome.ad.jp/kegg/pathway.html). Haploview (www.broad.mit.edu/mpg/haploview) was used to select tagging SNPs in the ACE gene from the HapMap project data.
Genotyping was performed (without prior knowledge of patients' grouping) using standard polymerase chain reaction technique to detect the insertion/deletion (IN/DEL) variant of the ACE gene (rs4646994) (28). All other SNPs were genotyped by real-time polymerase chain reaction using the ABI 7000 sequence detection system (Applied Biosystems, UK) and TaqMan chemistry in predesigned and validated TaqMan SNP genotyping assays (Applied Biosystems, Warrington, UK) (https://www2.appliedbiosystems.com). In total, six SNPs were genotyped; four in the ACE gene (rs4646994, rs4267385, rs4611524, rs4968591) and one in each angiotensinogen (AGT) (rs699) and angiotensin II type I receptor (AGTR1) (rs5186).
Catabolic Studies
Protocols were used as previously described (13). Briefly, Aprotinin (Bayer New Zealand Ltd, 0.5 mg) radiolabeled with technetium (approx 80MBq) (Apr*) was injected intravenously. Renal activity was measured (19–26 hr) from kidney imaging using a large field of view twin head gamma camera (SMV Vision DST-XL, linked to a Nuclear Diagnostics, imaging computer system). Timed urine collections (over 26 hr) and radioactive counting were made using a gamma counter (Packard Cobra Auto Gamma sample counter, GMI, LA).
Urinary Markers of Injury
Urinary pH, ammonia/titratable acidity, and total N-acetyl-β,d-glucosaminidase (NAG) activity (24 hr urine) were measured as previously (13, 16, 17). Monocyte- chemoattractant protein (MCP)-1 and TGF-β1 were measured using specific Quantikine Human enzyme linked immunosorbent assay kits respectively (R&D Systems, Abingdon, UK).
Other Investigations
Plasma renin and aldosterone activity were measured using solid-phase radioimmunoassay and Berthold LB 2111 gamma counter. Calcineurin-inhibitor trough levels were measured using high performance liquid chromatography and tandem mass spectrometry. Plasma glutathione (29, 30), glutathione peroxidase (31), vitamins A/E, β carotene, lycopene, and α-tocopherol and selenium levels (32, 33) were measured as previously in our laboratories.
STATISTICAL METHODS AND DATA ANALYSIS
All results and analyses are presented on an “intention-to-treat” dataset at endpoint. No interim analysis was permitted. Statistical analyses were performed using SPSS and SigmaPlot for Windows (SPSS Inc, Chicago, IL). All results are expressed as arithmetic means±SE unless otherwise stated, with 95% confidence intervals (CIs) and effect size as appropriate. In individual patients, paired Student's t tests were used with Tukey's modification to adjust for multiple comparisons.
Statistical analyses on nonparametric data were performed after logarithmic transformation and expressed as geometric means (SE). Comparisons of GFR methodologies were assessed using linear regression and correlation coefficients.
Proteinuria, a variable important to outcome measures, was significantly different in the two groups. Thus, a baseline-adjusted analysis of covariance was applied. This allowed comparison of changes from baseline to end of treatment (after 1 year) taking into account the primary markers as factors and baseline proteinuria as covariate.
Handling of “Missing” Data Points
Methods of dealing with this were decided a priori. Missing data rates were calculated and scrutinized and classified as “missing at random.” Since there are no universal strategies of dealing with missing data, multiple approaches were taken, including using closest match missing data (34), best and worst case scenario, imputation of the mean, and last observation carried forward (35). Listwise deletion was not used, because this does not represent the costs incurred and fails to make maximum use of all available data and would result in major loss of statistical power (36). There were no significant differences observed between these methods, and therefore, data are presented using imputation of the group mean (37).
Genotyping Statistical analysis
StatsDirect version 2.6.2 and Haploview version 3.32 statistical software programs were used to calculate allele, genotype, and haplotype frequencies using contingency tables, and Minitab 15 software used to perform analysis of variance analysis. In addition, categorical data analysis and continuous data analysis were applied.
Renal Catabolism of Radiolabeled Aprotinin
Data were handled as previously described (13).
RESULTS
Clinical Parameters
Patients (Consolidated Standards of Reported Trials)
Forty-seven patients were recruited; 25 randomized into group A (lisinopril treated) and 22 in group B (control patients). In group A, 17 completed the protocol, and eight were withdrawn (reasons stated Fig. 1). In group B, 17 completed the study, and five patients did not. One patient required transfusion postbiopsy.
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FIGURE 1.:
Patient flow diagram.
Demographics and Clinical Details
The groups were comparable except for a preponderance of deceased donors to live-related grafts in group B. Immunosuppression was similar; all but five patients in group A and six in group B were on ciclosporine-based immunosuppression (the rest were on prednisolone with azathioprine or mycophenolate mofetil), and one patient in group B was on tacrolimus (Tables 1 and 2).
TABLE 1: Demographic data of kidney donors and renal transplant patients
TABLE 2: Immunosuppression, patient and graft survival, and hematological parameters in renal transplant patients
All patients were taking at least one hypotensive agent (average 2–4) (β-blockers, vasodilators, or calcium-channel blockers±diuretics). The mean arterial blood pressures (diastolic+1/3[systolic-diastolic]) were suboptimal in most patients, but comparable at baseline, 3, 6 months, and end of the study in both groups (Table 3, Fig. 2a). Plasma potassium and bicarbonate levels were normal throughout (see Table, Supplemental Digital Content 2, https://links.lww.com/TP/A123). Group A required more bicarbonate, 8.5±1.0 g/day (0.11±0.01 mg/kg/day, 0.02–0.34 mg/kg/day) (95% CI 6.5–10.5) versus 4.5±1.0 g/d (0.05±0.007 mg/kg/day, 0.02–0.17) (95% CI 2.5–6.5) in group B, (F [2,43]=4.78, P<0.05, effect size, ω=0.37; Figure 2a). Hemoglobin levels and erythropoietin use were similar (Table 2).
TABLE 3: Changes in measurements of renal glomerular hemodynamics
FIGURE 2.:
Blood pressure control in the individual patient, a(i). Group A (lisinopril-treated) black lines; Group B (controls) grey lines (a); Systolic blood pressure (SBP) ┈ dashed lines; diastolic blood pressure (DBP) ..... dotted lines; and mean arterial blood pressures (MAP in mmHg) — solid lines (ii) Dose of oral sodium bicarbonate (g/day) given to patients in Group A (black line) and Group B (grey line) to maintain normal plasma potassium and bicarbonate levels throughout year of study. Renal graft function in the individual patients (b–e). Glomerular filtration rate (GFR), 51CrEDTA clearance (ml/min/1.73 m2) (b), plasma urea (μmol/L) (c), plasma creatinine (μmol/L) (d) and (e) proteinuria (g/24 h) in GroupA – the lisinopril-treated patients and Group B (control) patients during the year of study.
Histopathology
Renal biopsies were performed in 34 patients (>70%). Of the 13 patients not biopsied, seven were in group A and six in group B. Renal biopsy was not possible in all patients because of anxiety (six), learning difficulties (2), low platelets (1), hydronephrosis (long-standing, 1), and patients withdrawn from the trial (3). A senior renal pathologist blinded to group allocation analyzed biopsies. Most had grade I or II CAN (see Table, Supplemental Digital Content 3, https://links.lww.com/TP/A124) with a preponderance of grade I in group A and grade II in group B. None of the patients were treated empirically for “acute rejection.”
Graft Function and Progression Rates
51CrEDTA clearance (GFR) was similar at baseline and throughout in both groups (Table 3). At baseline, GFR in group A was 25.0±1.6 mL/min/1.73 m2, 11.7 to 47.6 mL/min/1.73 m2 (three ≤15, two 31–59, rest ≤30) and in group B 30±2.9 mL/min/1.73 m2, 12.0 to 58.2 mL/min/1.73 m2 (three ≤15, six 31–59, rest <30). Likewise, the rate of decline of graft function after 1 year was similar; −4.8±3.0 mL/min/year/1.73 m2 in group A and −2.3±1.7 group B. Figure 2b illustrates the results in the individual patients. In all patients with more than 30% reduction in proteinuria after 1 year, graft progression was −2.4±2.4 mL/min/year/1.73 m2 versus −6.7±1.8 in the rest (P=0.17). 51CrEDTA clearances correlated poorly with CrCl measurements or estimated GFR (see Figure, Supplemental Digital Content 4, https://links.lww.com/TP/A125). Graft or patient survival was similar in both groups (Table 2).
Proteinuria
Proteinuria was significantly greater, at baseline, in group A patients (Table 3), averaging 2.8±0.4 g/24 hr (0.9–7.2 g/24 hr; one patient 0.9, nine 1–2, twelve >2–5, and three >5g/24 hr) compared with group B patients 1.7±0.2 g/24 hr (0.6–4.8 g/24 hr; four 0.6–0.9, 13 >1–2, and five >2–5 g/24 hr), P<0.01. Two patients in group A had coexisting recurrent/de novo IgA nephropathy. After 1 year, proteinuria was reduced only in group A to 1.8±0.4 g/24 hr, (F [2,43]=5.51, P<0.01, ω=0.46), with more than or equal to 30% reduction in 11 patients (22.8±11.4, 33.3–95.0%). Response was maximal in patients with greater proteinuria. Figure 2e illustrates the results in the individual patients. There was a lag of 1 to 2 years between proteinuria reaching 1 g/24 hr and an apparent deterioration in graft function (see Table, Supplemental Digital Content 5, https://links.lww.com/TP/A126), but the time lag was shorter in group A.
Genotyping
All 47 patients were successfully genotyped, but full data sets at 6 months follow-up were available for 19 in group A and 18 in group B. These patients were then included in further analyses. Minor allele frequencies for the six variants genotyped in our study were similar to the reported frequencies in the dbSNP public database (see Table, Supplemental Digital Content 6, https://links.lww.com/TP/A127). There was no association of polymorphisms in the three genes tested with GFR or with a reduction in proteinuria in group A patients. However, analysis of all patients with more than 30% reduction in proteinuria regardless of therapy (n=21/37), showed a significant association with rs699 polymorphism in AGT using both categorical (odds ratio 3.1, Pc=0.02, 95% CI 1.12–8.8) and continuous data (DF2, F=3.41, P=0.04) analyses (Fig. 3a, b).
FIGURE 3.:
Genotyping in the renal transplant recipient patients. (a) PCR method used to detect IN/DEL polymorphism in the ACE gene. Electrophoresis gel using two sets of primers in two PCR reactions. Reaction 1 produced a 490 bp fragment in insertion allele (II) homozygous individuals, a 190 bp fragment in deletion allele (DD) homozygous individuals and both 490 bp and 190 bp in ID heterozygotes. In order to prevent mistyping of ID individuals due to the preferential amplification of the D allele, reaction two used insertion specific primer pair to verify each DD genotype. In the presence of the I allele, PCR two produced a 335 bp product. (b)(i) Box-plot diagram of the relationship between proteinuria measured over one year in the presence or absence of lisinopril treatment and the AGT rs699 genotype. The number of patients in three genotype groups: AA=11; AG=22; and GG=6. The highest increase in proteinuria was detected in GG (threonine) homozygous individuals (p<0.04, 95%CI). (ii) The individual value plots of the relationship between proteinuria the AGT and AGTR1 genotypes in all patients at least six months after randomisation. The AA genotype in AGT rs699 was associated with the lowest proteinuria levels (p<0.01) (ie patients with the best prognosis are the A allele carriers) (iii) The CC genotype in AGTR1 rs5186 was associated with the highest proteinuria levels (p<0.001), however only three individuals were carriers of the CC genotype. (c) Proximal renal tubular catabolism of radiolabelled aprotinin in the individual patients before and after treatment with lisinopril. (i) Kidney uptake (% of dose), (ii) metabolism (% of dose/h). (d) Urinary pH, (e) ammonia (mmol/h) and (f) titratable acidity (mmol/h) in the individual patients in groups A and B during the year of study.
Apr* Catabolic Studies
Apr* catabolic studies were not possible for the first five patients because only ethical approval to use aprotinin was not yet available. Kidney uptake of Apr* fell progressively with time from 21.2±1.2 at baseline (95% CI 18.8–23.6; n=21) to 14.3±1.1% of dose at 12 months (95% CI 12.1–16.5; n=12), (F [2,22]=12.45, P<0.001, ω=0.24) (see Table, Supplemental Digital Content 7, https://links.lww.com/TP/A128). Likewise, the rate of appearance of the free urinary pertechnetate over 26 hr fell from 0.94±0.04 at baseline (95% CI 0.87–1.02) to 0.71±0.03% of dose/hr after 1 year (95% CI 0.65–0.77) (F [2,22]=4.77, P<0.05, ω=0.21). Fractional degradation of Apr* was unchanged after 1 year. Figure 3c illustrates the results in individual patients. By 1 year, kidney uptake fell in all but two patients (unchanged). Metabolism of Apr* fell in all but four patients (unchanged).
Plasma/Urine Biochemistries and Markers of Injury
Plasma aldosterone levels (higher than the reference range) fell significantly after 1 year only in group A. The 24-hr urinary urea excretion (reflecting dietary protein intake and acid-load) were comparable (68.3±3.9 group A and 62.4±4.3 g/day group B). PTH values were less than 21 pmol/L in both groups. The 24-hr urinary sodium excretion was higher in group A patients at baseline and after 1 year. The 24-hr urinary potassium excretion (comparable at baseline) was higher in group A patients after 1 year. Plasma markers of oxidative stress were comparable and unchanged after 1 year (see Tables, Supplemental Digital Content 2 and 8, https://links.lww.com/TP/A123 and https://links.lww.com/TP/A129).
Urinary pH, ammonia, and titratable acidity were similar at baseline. Figures 3d to f illustrate results in individual patients. Urinary pH increased progressively with time in group A patients only (P=0.051). Both ammonia excretion and titratable acidity were reduced over the year in group A patients. In group B, only ammonia excretion fell. Values for NAG, MCP-1, and TGF-β1varied with overlap in both groups. Total 24-hr urinary NAG was significantly higher at baseline in group A patients but did not change with time. Urinary MCP-1 and TGF-β1 were comparable at baseline. Only TGF-β1 activity was greater in group A patients after 1 year.
DISCUSSION
CAN represents a syndrome rather than a single disease entity, characterized histologically by progressive vascular obliteration, glomerulosclerosis, TIF, and tubular atrophy (2, 5). This heterogeneity was reflected in our patient population. It was not possible to biopsy 13 patients for the reasons stated, and we acknowledge this limitation. However, there were equal numbers of patients not biopsied in both patient groups, and they fulfilled all other inclusion criteria and particularly persistent proteinuria and chronic graft dysfunction. In addition, none of these patients had calcineurin-inhibitor toxicity. Moreover, data were also entirely comparable when reanalyzed to include only the 34 patients with biopsy-proven CAN (see Table, Supplemental Digital Content 9, https://links.lww.com/TP/A130). The pathogenesis of CAN remains poorly understood. The complex combined effects of hypertension, reduced nephron mass, and proteinuria are important nonimmunologic causes (16). Why group B patients with less proteinuria had more severe chronic interstitial fibrosis remains unexplained but sampling or small numbers may be factors.
A reduction in the GFR in the first year posttransplantation is a strong predictor of subsequent graft loss (38–40). Decline of graft function thereafter is not linear and slower than in native nephropathies. Our data concur with the reported rates of decline of graft function, which vary between 2 and 5 mL/min/year averaging overall at 2 mL/min/year (2, 38–40). In a review of a more than 40,000 renal transplant recipients the annual rate of decline of graft function was estimated to be −1.66±6.51 mL/min/year (41). Isotopic measurements of GFR are the “gold” standard for evaluating renal graft function. We, like others (42, 43), found poor correlations between GFR measured isotopically and other methods. Differences in CrCl between the two groups reflect the inaccuracies of this method especially in patients with poor GFR. Lisinopril treatment for one year had no measured adverse effect on the rate of decline of graft function, but there was no amelioration either. Our data were limited to 1 year, and RCTs more than 3 to 5 years are needed. By contrast, Hiremath et al. (10) (systematic review) concluded a significant GFR deterioration of −5.8 mL/min attributed to the use of ACE inhibitors in renal transplantation However, the RCTs included (21) had varying protocols and methodologies, and in particular, few trials with long follow-up (in 12 <6 months) or studies commencing many years posttransplantation. Sample size was small (median of 31), and GFR was often not measured isotopically. Proteinuria was only reported in four trials, and CAN was not specifically addressed (nor metabolic acidosis or peptide/protein hypercatabolism). Retrospective studies and small observational studies have indicated positive effects of ACE inhibitors on graft survival (and proteinuria) independent of blood pressure control (11, 13, 15, 44–46).
Progression to ESRF correlates best with the extent of TIF (47). In diseased native kidneys, proteinuria and progression to ESRF are often linked (6–8, 48). This is also true in patients with CAN, where proteinuria and hypertension are frequently present (2, 12, 13, 16, 49, 50). In the Spanish CAN study (>3000 patients), graft survival was significantly lower in proteinuric patients and an independent risk factor for cardiovascular disease and mortality (12). A decrease of 0.1g/day in proteinuria has been associated with a 12% reduction in renal graft loss (49). Studies support a direct pathogenic role of proteinuria in the proximal tubules increasing oxidative metabolism and triggering mediators of tissue injury promoting TIF (80, 13, 16–19, 51–53).
As in other CAN studies (11, 13–15), lisinopril significantly reduced proteinuria in group A. There were differences in the effects of lisinopril treatment on proteinuria in the individual patients relating to variations in sodium balance, blood pressure, use of diuretics, and genetic factors (discussed below). Differences in proteinuria between the two groups of patients occurred because of baseline proteinuria more than 5.0 g/24 hr in three patients in the lisinopril-treated group (two patients had coexisting recurrent/de novo IgA nephropathy). When data from these three patients are excluded from the analysis, the average baseline proteinuria in the two groups is not statistically different. A greater renoprotective effect of lisinopril might have been masked because of differences in baseline proteinuria. The interval between proteinuria reaching 1 g/24 hr and first apparent deterioration in renal graft were shorter in group A, and this despite a preponderance of live-related transplants in this group (these tend to be superior in terms of long-term graft survival). These data suggest faster rates of progression in group A patients before commencing lisinopril. In all patients, proteinuria predated the changes in graft function by 1 to 2 years. This observation has not, to our knowledge, been previously described.
Hypertension is a critical factor adversely affecting graft survival after the first year independent of baseline renal function, and increases proteinuria and cardiovascular disease (54–57). Our patient population, as in other studies (57) had less than optimal blood pressure control (compared with a mean arterial blood pressure of <97 mm Hg), but without significant differences between the groups. Controlling systolic blood pressure to less than or equal to 140 mm Hg during the first 5 years posttransplantation is associated with the best graft and patient outcomes (57). However, this is difficult in patients on calcineurin inhibitor-based therapy.
We like others (58) found ACE inhibitors safe to use in transplant recipients. We did not, unlike others (10, 15), observe increased anemia (or erythropoietin use) secondary to lisinopril treatment. However, patients were sensitive to cough and three developed renal artery stenosis. There were other dropouts (reasons stated), but this was anticipated during the planning of the trial. However, in pilot studies needed to design this RCT (11, 13), lisinopril increased metabolic acidosis (with life-threatening hyperkalaemia). We, therefore, prophylactically treated metabolic acidosis in all patients with bicarbonate therapy. Reducing dietary sodium intake avoided aggravation of hypertension risking pulmonary congestion, especially in patients with comorbid cardiovascular disease. Metabolic acidosis, even without ACE-inhibitors, is highly prevalent in renal transplant recipients (in >60%) (24), and results from (1) inability to maintain acid-base balance as graft function declines; (2) renal tubular acidosis (secondary to the effects of excess PTH, which was not a major concern in our patients); and (3) the effects of calcineurin inhibitors on the tubules (most patients were on ciclosporine).
ACE inhibitors increase metabolic acidosis by (1) directly reducing renal ammoniagenesis, the primary buffer produced predominately by the proximal tubules, (2) indirectly by reducing protein delivery to the proximal tubules (as in group A patients), and (3) by disrupting aldosterone effects on potassium and hydrogen ion exchange in the distal tubule (59). In group A, only plasma aldosterone was reduced after 1 year and 24-hr urinary potassium excretion was greater than in group B. In other studies using ACE-inhibitors, plasma aldosterone levels were reduced only short term and hyperkalaemia persisted (60), but bicarbonate was not given.
The renal handling of low molecular weight peptides may be similar to that for large proteins (61). Aprotinin (MW 6,500 Daltons) has a high affinity for renal proximal tubules and is catabolized in the tubules. Renal catabolic studies were not carried out in group B patients because of prohibitive costs. Lisinopril treatment for 1 year led to a significant reduction in Apr* catabolism in parallel with the reduction in proteinuria and urinary ammonia in group A patients. Apr* renal tubular uptake was comparable quantitatively with our pilot data (13). However, unlike before, we were unable to demonstrate a significant amelioration of urinary markers of injury, inflammation or fibrosis after lisinopril. However, this is not so surprising, because there may have been a “masked” benefit from the onset derived from bicarbonate supplementation (18) given to all patients. Others have shown a reduction in NAG and TGF-β1 in patients after angiotensin II antagonists (62, 63).
We found no association either with genetic polymorphisms in the three genes ACE, AGT, and AGTR1 in response to lisinopril on graft function. However, GFRs were similar in both groups after 1 year, and the population observed was small. The DD (homozygous for deletion) genotype has been linked with a poor response to ACE-inhibitors therapy, M235T (rs699) and AGT and AGTR1 gene polymorphism with progressive nephropathies (64–67). Nevertheless, changes in proteinuria over 1 year in all our patients seemed in part at least to be determined by genetic variants in the ACE pathway (AA genotype in the AGT rs699 polymorphism). This association has previously been linked with a better prognosis for allograft survival in CAN.
CONCLUSION
The rate of decline of renal graft function in patients with CAN and severe renal impairment was not adversely affected by lisinopril therapy given for 1 year, but there was no amelioration either. However, lisinopril significantly reduced proteinuria, renal proximal tubular polypeptide catabolism, plasma aldosterone, and ammonia excretion. These data together suggest relative preservation of graft function, and this despite increased baseline proteinuria compared with control patients. Treating metabolic acidosis allowed safe and prolonged use of ACE-inhibitors. Large multicenter RCTs more than 3 to 5 years are needed to evaluate the efficacy of ACE inhibitors on renal graft function and progression.
ACKNOWLEDGMENTS
The authors thank Mr A. Bakran for allowing to recruit some of his patients into this study. They also thank acknowledge Yi Ning Chiang, a Medical student from the University of Liver pool who helped with genotyping during her elective period in the University Department of Pharmacology.
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