Angiotensin-Converting Enzyme Genotype and Renal

Transcription

1 Angiotensin-Converting Enzyme Genotype and Renal Allograft Survival JOACHIM BEIGE,* SABINE SCHERER,t ANGELA WEBER,* STEFAN ENGELI,* GERD OFFERMANN,* GERHARD OPELZ,t ARMIN DISTLER,* and ARYA M. SHARMA* *Departrnent of Internal Medicine, Division of Endocrinology and Nephrology, Universit#{228}tsklinikum Benjamin Franklin, Free Universily of Berlin, Berlin, Germany; and Institute of Immunology, Department of Transplantation Immunology, Ruprecht-Karls-Universitat. Heidelberg, Germany. Abstract. Increased activity of the renin-angiotensin system has been implicated in decreased, long-term survival of renal allografts. Recent studies suggest that a deletion variant of the angiotensin-converting enzyme, associated with increased humoral and tissue activity of this enzyme, is a risk factor for the development of diabetic nephropathy and the progression of IgA nephropathy. To determine whether the deletion variant of the angiotensin-converting enzyme gene influences the longterm outcome in renal transplant recipients, the relationship between donor and recipient angiotensin-converting enzyme genotype and clinical outcome were examined over a follow-up period up to 3 mo in a cohort of 269 Caucasian patients undergoing kidney transplantation between 1988 and In a subsequent case control study, the frequencies of the angiotensin-converting enzyme genotype were compared in a group of Caucasian patients with a graft survival of less than 3 yr (mean survival, 1 1 mo; n = 328) with the frequencies in patients with a graft survival of at least 3 yr (mean survival, 65 mo, n = 461). Neither in the cohort nor in the case control study was there a significant effect of recipient or donor angiotensin-converting enzyme genotype on transplant survival. Furthermore, the frequency of the angiotensin-converting enzyme deletion allele both in recipients and donors was similar to that reported in Caucasian controls. This study, therefore, does not support the hypothesis that the recipient or donor angiotensin-converting enzyme insertion/deletion polymorphism is an important determinant of transplant survival in Caucasian patients undergoing renal transplantation. (J Am Soc Nephrol 8: , 1997) Despite considerable improvement during the last two decades in early graft survival, a gradual decrease in graft function over time remains a severe obstacle to the long-term success of kidney transplantation. The mechanisms leading to the progressive loss of graft function, often termed a chronic rejection or chronic allograft injury, remain largely unknown, and no effective therapy is available (1). Several lines of evidence suggest a role for the renin-angiotensin system in the development and progression of chronic allograft injury. Hypertrophy of the juxtaglomerular apparatus is a common histological feature of chronic rejection and presumably indicates increased renin secretion (2). Higher plasma renin activity has been reported in patients with worsening of graft function (3) and has also been implicated as a cause of post-transplant hypertension (4), an important risk factor for allograft injury (1). Serum and tissue levels of angiotensin-converting enzyme (ACE), the key enzyme for the generation of angiotensin II Received November 7, Accepted January 27, Correspondence to Dr. Arya M. Sharma, Medizinische Klinik, Klinikum Benjamin Franklin, Free University of Berlin, Hindenburgdamm 3, D-122 Berlin, Germany / $3./ Journal of the American Society of Nephrology Copyright ( 1997 by the American Society of Nephrology from angiotensin I, are now known to be under genetic control. Thus, individuals harboring a deletion (D) of a 289-bp Alu sequence located in intron 16 of the gene display higher humoral and tissue activity of this enzyme than individuals who are homozygous for the insertion (I) allele (5,6). Although ACE exists in a free circulating form, most of it is expressed as a transmembrane protein that is also present both in endothelial and epithelial cells in the kidney (7). Apart from its role in the renin-angiotensin system, ACE is also the major inactivating enzyme of bradykinin, substance P, and other peptides that play important roles in several aspects of inflammatory and immunological responses (8). Importantly, large amounts of ACE are expressed constitutively in macrophages (9), monocytes, and T lymphocytes (1), and in the latter, ACE expression is under genetic control, with homozygotes for the D allele expressing markedly higher levels of this enzyme than other individuals (1). Recent studies have now implicated the ACE-D variant as a risk factor for the development of diabetic nephropathy (I 1-14) and an increased rate of progression of IgA nephropathy (15-17), but these findings have not been confirmed by all investigators (18-22). The aim of the present study, therefore, was to determine the relationship between the ACE genotype and renal allograft survival in patients undergoing cadaveric renal transplantation for terminal renal insufficiency. The systemic activity of the renin-angiotensin system depends on

2 I 32 Journal of the American Society of Nephrology recipient genotype, and graft survival also may be influenced by ACE activity within the allograft. Therefore, both donor and recipient genotypes were examined. Materials and Methods Consent for collection of DNA samples and permission to review the medical records regarding outcome variables were obtained within the setting of the International Collaborative Transplant Study (23). Genotvping Donor and recipient genomic DNA was prepared from peripheral white blood cells, using routine DNA extraction techniques. Subsequently. the ACE l/d variant region was amplified with the PCR technique, using a flanking primer pair as described previously (5). To exclude mistyping of heterozygotes, all DD individuals were also examined using primers specific for the insertion variant as described previously (24). Amplicons were visualized on 2.5% ethidium bromide-stained agarose gels. Cohort Study DNA samples were collected prospectively between 1988 and 1993 from 269 consecutive individuals receiving kidney transplants at the Universit#{228}tsklinikum Benjamin Franklin in Berlin and their respective donors. The clinical course for the first two and a half years (3 mo) after transplantation was analyzed by blinded review of all case records with regard to both graft survival and occurrence of acute and chronic graft rejection. Core needle graft biopsy was performed in all instances of a significant increase in serum creatinine. Chronic graft rejection was defined as a 2% elevation of serum creatinine from baseline levels (determined as the average of at least three stable values) at least 3 mo after transplantation and the demonstration of vascular sclerosis in the graft biopsy. Acute rejection was defined as an accelerated rise in creatinine requiring the initiation of rejection therapy, and in most cases was also confirmed by transplant biopsy. Case Control Study To compare allele frequencies of the ACE gene polymorphism in patients with early transplant loss versus long-term survival, we examined 789 Caucasian patients undergoing first-time transplantation of cadaver kidneys from centers in North America and Western Europe who had been recruited for the International Collaborative Transplant Study (23) between 1988 and Patient selection was based on the ftllowing criteria: ( ases: patients with a graft survival of less than 3 yr (a = 328); Controls: patients with a graft survival of at least 3 yr (a = 46 1 ). Patients with graft survival of less than 3 d were not recruited for the study. ACE genotype was also determined for all donors. All data and follow-up information were recorded within the framework of the Collaborative Transplant Study. Statistical Analyses In the cohort study, graft survival and the occurrence of chronic rejection were analyzed by Kaplan-Meier life-table analysis and twotailed global log-rank testing. In the case control study, allelic frequencies in the different groups were compared by statistics. Results Cohort Study Genotype distribution in recipients (II = S 1, ID = 149, DD = 69, frequency of D allele [qd] =.53) and donors (II = 55, ID = 134, DD = 8, qd =.55) was similar, and allele frequencies were comparable to those described for Caucasian populations by other investigators (5,6). Gender, body mass index, recipient and donor age, number of hypertensive patients, and antihypertensive agents, as well as diseases leading to transplantation, were not significantly different among the various genotypic groups (Table I ). Transplant survival as analyzed by the Kaplan-Meier method was not significantly affected by either recipient or donor ACE genotype (Figure 1). Causes of graft loss likewise were not significantly different among the various genotypic groups (Table 2). Similarly, genotype distribution in all patients diagnosed by renal biopsy as having chronic graft rejection was similar to that observed in the entire cohort (II = 1, ID = 42, ID = 1 1, qd = 51), indicating no association between chronic graft rejection and the ACE-D allele. Table I. Patient characterization by recipient ACE genotype in the cohort study (mean ± SD)t Characteristic II (it = 51) ID (n = 149) DD (n = 69) Recipient gender (F/M) 14/14 64/85 27/42 BMI (kg/m2) 21.8 ± ± ± 3.5 Recipient age 42.9 ± ± ± 2.2 Donor age 39.3 ± ± ± 15.8 Number of hypertensive patients 27 (54%) 91 (62%) 43 (62%) Number of antihypertensive agentsb 1.9 ± ± ±.7 Diseases leading to transplantation chronic glomerulonephritis diabetic nephropathy polycystic kidney disease analgesic nephnopathy others a ACE, angiotensin-converting enzyme; I, insertion; D, deletion; F, female: M, male: BMI. body mass index. h Ii patients treated for hypertension.

3 ACE Genotype and Renal Graft Survival 1321 Recipient Genotype Donor Genotype > U) ) numbers at risk #{149}ll 51 AID 149 #{149}DD months II.- :::- Ce >? 5 I) numbers at risk. II A ID 134 #{149}DD months Figure 1. Relationship between graft survival and recipient (left panel) and donor (right panel) ACE genotype. Table 2. Causes of graft loss by recipient ACE genotype in the cohort studya Characteristic II (n = 51) ID (ti = 149) DD (n 69) Cause of graft loss in surviving recipients acute injury followed by graft loss chronic rejection other S (1%) 9 (18%) 27 (18%) 18 (12%) 2(1%) 7 (1%) 7 (1%) Subtotal Cause of recipient death with maintained graft function cardiovascular nonrenal carcinoma infectious and inflammatory diseases others unknown 14 (27%) I (2%) 2 (4%) 1 (2%) 47 (27%) 6 (4%) 5 (3%) 1 ( 1 %) 2 (1%) 3 (2%) 14 (2%) 4 (7%) 5 (7%) 1 (1%) 3 (4%) Subtotal 4(8%) 17(11%) 13 (19%) Total number of graft losses 1 8 (36%) 64 (43%) 27 (39%) a Abbreviations as in Table 1. Case Control Study Mean graft survival in the recipients with early graft loss (cases) was 1 1 mo (range, 7 d to 36 mo), whereas the mean follow-up period in patients with graft survival beyond 3 yr (controls) was 65 mo (range, 36 to 97 mo). Recipient ACE genotype distribution in cases (II = 69, ID = 16, DD = 99, qd =.55) and controls (II = 94, ID = 228, DD 139, qd.55) and donor genotype distribution in cases (II = 68, ID = 172, DD = 88, qd =.53) and controls (II = 87, ID 237, DD = 137, qd =.55) were similar and comparable to those in the cohort study. Recipient gender and age, donor age, donor genotype, and diseases leading to transplantation were not significantly different between the three genotypic groups (Table 3). Discussion Neither in our prospective cohort study nor in our large case control study were we able to detect a significant influence of the ACE genotype on transplant survival. In the cohort study, life-table analysis revealed similar transplant survival over the 3-mo follow-up period regardless of donor or recipient genotype. Similarly, neither genotypic distribution nor allelic fre-

4 I 322 Journal of the American Society of Nephrology Table 3. Patient characterization by recipient ACE genotype in the case control study (mean ± SD)d Characteristic Cases Controls II (it = 69) ID (n 16) DD (n = 99) II (it = 94) ID (n = 228) DD (n = 139) Recipient gender (F/M) 29/4 56/14 45/54 4/54 12/126 62/77 Recipient age 45. ± ± ± ± ± ± 12.8 Donor age 35.4 ± ± ± ± ± ± 15.3 Donor genotype (IIIIDIDD) 8/44/17 32/83/45 28/45/26 15/49/3 42/122/64 3/66/43 Diseases leading to transplantation chronic glomerulonephritis diabetic nephropathy polycystic kidney disease analgesic nephropathy others a Abbreviations as in Table 1. quencies of the ACE genotype were markedly different between patients with early graft loss compared to the control group with grafts functioning over a mean follow-up period of 65 mo. Taken together, therefore, these data do not support the hypothesis that the ACE l/d polymorphism is an important determinant of renal allograft survival in Caucasian transplant recipients. The rationale for studying the relationship between the ACE genotype and renal allograft survival was provided by the fact that the renin-angiotensin system has been implicated in the pathogenesis of chronic rejection (1), the leading cause of graft loss in all genotypic groups in our study. The importance of the renin-angiotensin system for immune-mediated injury in renal (25), cardiac (26). and aortic (27) allografts in rats has been demonstrated previously by attenuation of injury by administration of ACE inhibitors. Also, short-term studies in man have demonstrated reduced proteinuria with ACE inhibition in hypertensive patients with renal transplants (28). Further rationale for the present study was provided by recent observations that the ACE-D allele may increase the risk of developing or may influence the rate of progression of other forms of chronic kidney disease. Several investigators have reported a higher frequency of the D allele in patients with diabetic nephropathy ( ), a finding that has not been confirmed by all investigators ( ). Furthermore, several recent reports showed an increased rate of progression in patients with IgA nephropathy harboring the ACE-D allele (15-17), an observation that also has not been confirmed by other investigators (22). In the only published report on the frequency of the ACE-D allele in renal transplant recipients thus far, Gaciong et al. (29) report a higher prevalence of the D allele (qd =.63) in transplant recipients versus healthy, age-matched blood donors (qd =.52). They suggest that this finding may be due to the fact that the D allele is a risk factor for the progression of renal disease. These observations, however, are not supported by our present data that the frequency of the D allele in renal allograft recipients both in the cohort and the case-control study is similar to that observed in healthy Caucasian controls in other studies (5,6). The lack of relationship between renal allograft survival and the ACE l/d genotype does not completely rule out a role for increased ACE activity in the development of chronic allograft rejection. Other, hitherto unrecognized or unstudied variants of the ACE gene (3) might be associated with either the development of end-stage renal failure or the progression of renal injury, including chronic transplant rejection, and the ACE gene therefore remains an attractive candidate gene. Furthermore, given that the development of chronic transplant rejection is probably the result of a complex interaction between a variety of immunologic, genetic, and environmental factors (1), our study does not rule out the importance of the ACE genotype in certain subsets of patients or under certain environmental conditions. Finally, because the ACE-D allele has been associated with increased humoral and tissue activity of this enzyme (5,6,1), it would be of interest to ascertain whether individuals with the DD genotype profit more from therapeutic blockade of the renin-angiotensin system than other individuals. This suggestion is based on the findings of Yoshida et al. (16), who demonstrated a significant reduction of proteinuria after ACE inhibition in patients with IgA nephropathy and the DD genotype, but not in patients with the ID or II genotype. Similarly, individuals harboring the D allele were found to have a larger decrease in BP during ACE inhibition than II patients (3 1), indicating that the ACE genotype may significantly influence the therapeutic response to blockade of the renin-angiotensin system. However, the present study does not support the hypothesis that the donor or recipient ACE-lID genotype is an important determinant of transplant survival in Caucasians undergoing renal transplantation for end-stage renal failure. Acknowledgments We thank Klaus Schlotter for technical assistance in analyzing the DNA samples. We are also grateful to Dr. Ulnike Schorr for expert

5 ACE Genotype and Renal Graft Survival 1323 help in preparing the manuscript. The study was supported in part by the Deutsche Forschungsgemeinschaft (Sh 35/2-2). References 1. Hostetter TH: Chronic transplant rejection. Kidney mt 46: , Crocker B, Ramos EL: Pathology of the renal allograft. In: Renal Pathology, edited by Tischer C, Brenner B, Philadelphia, Lippincott, 1989, pp Bresticker M, Nelson I, Huang C, Wolf J, Anderson B: Plasma renin activity in renal transplant patients with hypertension. Ant J Hyperteits 4: , Luke RG: Pathophysiology and treatment of posttransplant hypertension. J Am Soc Nephrol 2: , Rigat B, Hubert C, Alhenc Gelas F, Cambien F, Convol P, Soubnier F: An insertionldeletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Cliii Invest 86: , Tiret L, Rigat B, Visvikis S, Breda C, Convol P, Cambien F, Soubrier F: Evidence, from combined segregation and linkage analysis, that a variant of the angiotensin I-converting enzyme (ACE) gene controls plasma ACE levels. Am J Hum Genet 51: , Rosenberg ME, Smith LI, Correa Rotter R, Hostetter TH: The paradox of the renin-angiotensin system in chronic renal disease. Kidney mt 45: 43-41, Erdos EG: Angiotensin I converting enzyme and the changes in our concepts through the years [Lecture]. Hypertension 16: , Fniedland I, Setton C, Silverstein E: Induction of angiotensin converting enzyme in human monocytes in culture. Biochem Biophvs Res Co,n,nun 83: , Costerousse, Allegrini I, Lopez M, Alhenc Gelas F: Angiotensin I-converting enzyme in human circulating mononuclear cells: Genetic polymorphism of expression in T-lymphocytes. Biochem J 29: 33-4, Marre M, Bernadet P. Gallois Y, Savagner F, Guyene IT, Hallab M, Cambien F, Passa P. Alhenc-Gelas F: Relationships between angiotensin I converting enzyme gene polymorphism, plasma levels, and diabetic retinal and renal complications. Diabetes 43: , Mizuini 5, Hemmi H, Inoue A, Yoshikawa H, Tanegashima M, Fushimi T, Ishigami M, Amagasaki Y, Ohara T, Shimatake H: Angiotensin-converting enzyme polymorphism and development of diabetic nephropathy in non-insulin-dependent diabetes mellitus. Nep/iron 7: , Doi Y, Yoshizumi H, Yoshinari M, lino K, Yamamoto M, Ichikawa K, Iwase M, Fujishima M: Association between a polymorphism in the angiotensin-converting enzyme gene and microvascular complications in Japanese patients with NIDDM. Diabetologia 39: 97-12, Ohno T, Kawazu 5, Tomono 5: Association analyses of the polymorphisms of angiotensin-converting enzyme and angiotensinogen genes with diabetic nephropathy in Japanese noninsulin-dependent diabetics. Metabolism 45: , Yonioka T, Suehiro T, Yasuoka N, Hashimoto K, Kawada M: Polymorphism of the angiotensin converting enzyme gene and clinical aspects of IgA nephropathy. Clin Nephrol 44: 8-85, Yoshida H, Mitarai T, Kawamura T, Kitajima T, Miyazaki Y, Nagasawa R, Kawaguchi Y, Kubo H, Ichikawa I, Sakai : Role of the deletion of polymorphism of the angiotensin converting enzyme gene in the progression and therapeutic responsiveness of IgA nephropathy. J C/in Invest 96: , Harden PN, Geddes C, Rowe PA, Mcllroy JH, Boulton-lones M, Rodger RS, Junor BJ, Bniggs ID, Connell IM, Jardine AG: Polymorphisms in angiotensin-converting-enzyme gene and progression of IgA nephropathy. Lancet 345: , Schmidt 5, Schdne N, Ritz E: Diabetic nephropathy study group: Association of ACE gene polymorphism and diabetic nephropathy. Kidneylnt47: , Tarnow L, Cambien F, Rossing P. Nielsen FS, Hansen BV, Lecerf L, Poirier, Danilov S. Parving HH: Lack of relationship between an insertion/deletion polymorphism in the angiotensin I-converting enzyme gene and diabetic nephropathy and proliferative retinopathy in LDDM patients. Diabetes 44: , Chowdhury TA, Dronsfield MI, Kumar 5, Gough SLC, Gibson SP, Khatoon A, MacDonald F, Rowe BR, Dungen DB, Dean ID. Davies Si, Webben I, Smith PR, Mackin P. Marshall SM, Adu D, Morris P1, Todd IA, Barnett AH, Boulton Al, Bain SC: Examination of two genetic polymorphisms within the renin-angiotensin system: No evidence for an association with nephropathy in IDDM. Diabetologia 39: , Ringel I, Beige I, Kunz R, Distler A, Sharma AM: Genetic variants of the renin-angiotensin system, diabetic nephropathy, and hypertension. Diabetologia 4: , Fukushima T, Nomura 5, Kawai 5, Osawa G: ACE genotype and progression of IgA nephropathy [Letter]. Lancet 346: 57 1, Opelz G, Mytilineos I, Scherer S. Dunckley H, Trejaut I, Chapman I, Middleton D, Savage D, Fischer, Bignon ID, Bensa IC, Ekkehard A, Harriet N: Survival of DNA HLA-DR typed and matched cadaver kidney transplants: The collaborative transplant study. Lancet 338: , Lindpaintner K, Pfeffer MA, Kreutz R, Stampfer Ml, Grodstein F, LaMotte F, Buring I, Hennekens CH: A prospective evaluation of an angiotensin-converting-enzyme gene polymorphism and the risk of ischemic heart disease. N Engi J Med 332: , Rumble I: Comparison of effects of ACE inhibition with calcium channel blockade on renal disease in model combining genetic hypertension and diabetes. Am J Hvpertens 8: 53-57, Paul LC, Davidoff A, Benediktsson H: Cardiac allograft atherosclerosis in the rat: The effect of histocompatibility factors, cyclosponine, and an angiotensin-converting enzyme inhibitor. Transplantation 57: , Michel JB, Plissonnier D, Bruneval P: Effect of perindopril on the immune arterial wall remodeling in the rat model of arterial graft rejection. Am J Med 92: , Bochicchio T, Sandoval G, Ron, Perez Grovas H, Bordes I, Herrera Acosta I: Fosinopril prevents hypenfiltration and decreases proteinuria in post-transplant hypertensives. Kidney 1w 38: , Gaciong ZA, Religa P, Placha G, Rell K, Paczek L: ACE genotype and progression of IgA nephnopathy [Letter]. Lancet 346: 57, Villard E, Tiret L, Visvikis 5, Rakotovao R, Cambien F, Soubnier F: Identification of new polymorphisms of the angiotensin I-converting enzyme (ACE) gene, and study of their relationship to plasma ACE levels by two-qtl segregation-linkage analysis. Am J Hum Genet 58: , Hingorani AD, Jia HY, Stevens PA, Hopper R, Dickerson JEC, Brown Ml: Renin-angiotensin system gene polymorphisms influence blood pressure and the response to angiotensin converting enzyme inhibition. J Hypertens 13: , 1995