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1 Effects of Genetic Polymorphisms of Angiotensin in Children with Nephrotic Syndrome Yılmaz Tabel,* Afig Berdeli,# Sevgi Mir,* Erkin Serdaroglu,* Ebru Yılmaz* Keywords: RAS, Gene polymorphism, Nephrotic syndrome, Children *Department of Pediatric Nephrology, Ege University School of Medicine, Bornova, 35100, Izmir-Turkey #Laboratory of Molecular Medicine, Department of Pediatrics, Ege University School of Medicine, Bornova, 35100, Izmir-Turkey Correspondence to: Dr Yilmaz Tabel Inonu Universitesi, Turgut Ozal Tip Merkezi, Cocuk Sagligi ve Hast. AD 44280, Malatya/TURKEY Tel: Fax: yahoo.com Accepted for publication 23rd November 2005 JRAAS 2005;6: Peptidergic s Abstract Background. The renin-angiotensin system (RAS) has been considered to be responsible for the pathogenesis or progression of many diseases which may or may not be related to kidney. Genetic polymorphisms of the various components of the RAS have been associated with differences in the clinical course of several disease states in adults and children. Objectives. The purpose of our study was to investigate RAS gene polymorphisms in patients with steroid resistant primary focal segmental glomerulosclerosis (FSGS) and nephrotic syndrome responding to steroid therapy. Furthermore, we aimed to investigate whether there was an association between polymorphic alleles and responses to steroid therapy, the degree of renal dysfunction, and prevalence of end-stage renal disease (ESRD). Material and methods. One hundred and fifty-eight children with the diagnosis of nephrotic syndrome were recruited from the Nephrology unit in the Department of Paediatrics of Ege University. Forty-nine of them were classified as renal biopsy-proven FSGS and 102 patients were considered to have response to steroid treatment. Renal functional impairment was detected in 19 subjects with FSGS and end-stage renal insufficiency in 13 patients. The control group consisted of 287 healthy adult subjects. converting enzyme (ACE), angiotensin II type 1 receptor (AT 1 R) and angiotensinogen (AGT) gene polymorphisms were determined by the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique. Statistical significance was regarded as p<0.05. Results. There were no statistically significant differences for the C allele of AT 1 R or the T allele of AGT genes between the children with nephrotic syndrome and control group, but on the other hand statistically significant differences were detected for D allele of ACE gene. There was no significant relationship detected between the D allele of ACE, the C allele of AT 1 R or the T allele of AGT genes and response to steroid therapy, extent of renal dysfunction and the progression to ESRD. However, there was a significant relationship between T allele of AGT gene and resistance to steroid treatment (OR; 4,837, 95% CI; 1,723 13,577, p=0.01), renal dysfunction (OR; 3,189, 95% CI; 0,999 10,182, p=0.041) and progression to ESRD (OR; 3,852, 95% CI; 1,057 14,040, p=0.03). Conclusion. In this study, the angiotensino gene -235T allele was found to be related with steroid resistance, renal dysfunction and progression of ESRD in nephrotic syndrome. Introduction Idiopathic nephrotic syndrome is the most common cause of nephrotic syndrome in children. Two types of idiopathic nephrotic syndrome can be described according to the response to corticosteroids, steroid-responsive and steroid-resistant. Minimal change disease (MCD) most often responds to corticosteroids. A significant proportion of patients with focal segmental glomerulosclerosis (FSGS) respond to corticosteroids, whereas some patients with MCD are resistant to corticosteroids. 1-3 Moreover, stages of FSGS and MCD are distinguishable. Steroid resistant idiopathic nephrotic syndrome accounts for more than 10% of children who progress to end-stage renal disease (ESRD). Although the pathogenesis of ESRD is controversial, an altered renin angiotensin system (RAS) may play a role. Genetic factors have been suggested to be important in determining the progression of idiopathic nephrotic syndrome. Angiotensinconverting enzyme (ACE), angiotensinogen (AGT) and angiotensin II type 1 receptor (AT 1 R) are the potential candidates as prognostic factors in the progression of renal damage and the development of ESRD. 4 Angiotensin II (Ang II) is the most active product of the RAS, which is both a local and a systemic regulator of haemodynamics. 5 Ang II; which is active in promoting renal mesenchymal cell growth, in the synthesis and secretion of extracellular matrix, and in inflammatory events, plays a key role in progression of renal diseases. 6 RAS-related studies in children have particularly focused on nephrotic syndrome (NS) patients. 7,8 The polymorphism 138

2 Peptidergic s related to insertion or deletion of 287 bp part of intron 16 in the ACE gene is the first and the most commonly studied polymorphism related to RAS. 9 The other polymorphisms are A1166C polymorphism in AT 1 R and M235T polymorphism in AGT gene In this study, these three genes and the effects of their polymorphisms on clinical, laboratory, and histological findings in relation to response to treatment in childhood NS and progression to ESRD have been studied. Materials and Methods Definitions NS was diagnosed by oedema, hyperlipidaemia, serum albumin below 2.5 g/dl, and urinary protein excretion greater than 40 mg/m 2 per hour. The standard steroid therapy regimen consisted of prednisone 2 mg/kg per day (maximum dose 60 mg/day) in a single dose or two divided doses for 4 6 weeks, followed by alternate-day prednisone at a single dose for eight weeks, and including any tapering off period. Complete remission was accepted as urinary protein excretion less than 100 mg/m 2 per day for at least three consecutive days, partial remission as 100 1,000 mg/m 2 per day, and steroid resistance as urinary protein exceeding 1,000 mg/m 2 per day together with no clinical improvement for 8 10 weeks. A relapse was denoted by recurrence of nephrotic-range proteinuria requiring the reinstitution of daily prednisone therapy after the urine had been protein free for at least four weeks. A frequent relapse was defined as three or more relapses within 12 months in an initially steroid-responsive patient. Steroid dependency was defined as the tendency to relapse while on or during tapering of steroid dose, or within 14 days of steroid withdrawal. In the steroid resistant cases, 30 mg/ kg (maximum 1,000 mg) IV methyl prednisolone was used in six divided doses on alternate days as recommended by Mendoza et al. 18 At least six months oral cyclosporine A at 3 5 mg/kg/day and oral cyclophosphamide at 2.5 mg/kg/day for 12 weeks or at 3 mg/kg/day for 10 weeks were used. The histopathological diagnosis of FSGS and MCD was made according to histopathological criteria as described by the International Society for Kidney Disease in Children (ISKDC) for children with NS. 19 Hypertension was described as systolic and diastolic blood pressure above the 95th percentile for the appropriate age group and height on three consecutive measurements. 20 Glomerular filtration rate (GFR) was estimated using Schwartz formula 21 and compared to a standard clearance formula. Normal renal function was defined as GFR >80 ml/minute per 1.73 m 2, pre-dialysis chronic renal failure (CRF) as GFR ml/minute per 1.73 m 2, and ESRF as GFR <10 ml/minute per 1.73 m 2. Patients and Controls The study included 158 (93 male, 65 female) Caucasian Turkish nephrotic syndrome patients were recruited from the department of Paediatric Nephrology at the Ege University. The mean age at diagnosis was 3.53±2.87 years. The mean followup period was 4.6±3.7 years. All patients had presented with a full blown nephrotic syndrome. The Ethical Research Committee of the university approved the study. After informed consent for the genetic studies was obtained from the parents of all patients blood samples were obtained for DNA analysis. Exclusion criteria for the present study were as follows: presence of membranoproliferative glomerulonephritis, membranous nephropathy, congenital nephrotic syndrome and secondary nephrotic syndrome. A complete review was carried out for each patient, which included the patient s past and family history, physical examination, and data such as gender, age, height, weight, proteinuria, albumin levels, cholesterol and triglyceride levels, renal function tests, blood pressure on admission, treatment and response, number and time of relapses and renal biopsy findings. Two-hundred and eighty-seven ethnically matched unrelated healthy adult volunteers (156 males, 131 females and mean age was 29.1±11.8 years), without kidney diseases and hypertension served as a control group. This large group of healthy subjects enabled us to study the distribution of the various allelic polymorphisms of RAS in individuals of different ethnic backgrounds in Turkey. Analysis of ACE Genotype Genomic DNA was prepared from whole blood using QiAamp DNA Blood Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer s recommendations. A 50 ng sample of genomic DNA was amplified by PCR (Gene Amp PCR 9700, PE Applied Biosystems) in a 25 µl mixture of 1x Gene Amp PCR Buffer II with 1.5 mm MgCl 2 (Applied Biosystems, Brachburg, NJ, USA), 200 µm of each deoxynucleotide triphosphate (dntp) (Promega, Madison, WI, USA), 5 pmol sense and antisense primers, 0.2 µl AmpliTaq DNA polymerase (Applied Biosystems, 5 U/µl), and nuclease-free water. The sequences of the sense and antisense primers were 5 -CTGGAGACCACTCCCATCCTTTCT-3 and 5 -GATGTGGCCATCACATTCGTCAGAT-3 (TIB MOLBIOL Syntheselabor, Berlin, Germany), respectively. Thermal cycling was carried out at 94 o C for 10 minutes, then 32 cycles of 94 o C for 30 seconds, annealing at 58 o C for 50 seconds and extension at 72 o C for 60 seconds, followed by 7 minutes of final extension at 72 o C. The PCR products were separated by 2% agarose gel electrophoresis, and 490 bp with insertion (I) and 190 bp with deletion (D) were visualised with ethidium bromide staining in the UVP BioDoc-It Bio Imaging s (Upland, California, USA): To avoid the possibility of mistyping the ID heterozygotes as DD homozygotes, all DD 139

3 Peptidergic s samples were reamplified with a second primer pair specific for the inserted sequence intron 16 of the ACE gene. PCR was performed in a final 25 µl mixture of 1.5 mm MgCl 2, 20 pmol primers, 2 U Taq polymerase, and 100 ng genomic DNA. The primers for the inserted sequence were 5 -TGGGACCACAGCGCCCGCCACTAC-3 and 5 - TCGCCAGCCCTCCCATGCCCATAA-3 (TIB MOLBIOL Syntheselabor-BERLIN). Thermal cycling was carried out at 94 o C for 10 minutes, then 30 cycles with 30 seconds denaturation at 94 o C, followed by 30 seconds annealing at 67 o C, extension for 30 seconds at 72 o C, and 10 minutes final extension at 72 o C. The PCR products were separated by 2% agarose gel electrophoresis; the 335 bp DNA fragment with ethidium bromide staining was visualised in the UVP BioDoc- It Bio Imaging s. The I allele produced a 335 bp fragment, while no products were detected with the DD genotype, which assured the absence of mistyping. Analysis of AGT Genotype The AGT Met235Thr polymorphism was analysed according to Russ et al. 22 The T/C transition at nucleotide 704 (leading to a substitution of the amino acid Met to Thr) in exon 2 produces a half-site for the Tth111I restriction enzyme (Promega, Madison, WI). Introduction of the corresponding half site is achieved by using a PCR primer with two mismatches. These are located at positions 4 and 5 from its 3 end and do not interfere with elongation (reverse primer, 5 CAGGGTGCTGTCCACACTGGACCCC- 3 ; forward primer, 5 -CCGTTTGTGCAGGGCCTGGCTCTCT-3 ). (TIB MOLBIOL, Syntheselabor, Berlin-Germany). Amplification was carried out on a GeneAmp PCR 9700 (PE Applied Biosystems, Foster City, CA) in a 25 µl reaction mixture in 0.2 ml thinwall PCR strip tubes (Axygen Scientific, Inc., CA) containing 1 µl genomic DNA solution, GeneAmp PCR Gold Buffer (15 mmol/l Tris-HCl, ph 8.0, 50 mmol/l KCl; PE Applied Biosystems), 1.5 mmol MgCl 2, 50 µmol/l each of the dgtp, datp, dttp and dctp (Promega, Madison, WI), 5 pmol each forward and reverse primers and 1.0 U AmpliTaq Gold polymerase (PE Applied Biosystems). The PCR cycling conditions were as follows: an initial denaturation at 95 o C for 10 minutes; 35 cycles at 95 o C for 30 seconds, 68 o C for 45 seconds, and 72 o C for 1 minute; and a final extension at 72 o C for 7 minutes. The amplification yielded a PCR product of 165 bp. The PCR products for AGT gene was analysed on a 2.0% agarose gel prestained with ethidium bromide (0.5 µg/ml) for visualisation under ultraviolet light. Gels (15x15 cm) were run at 90 mv in 1xTBE buffer for 90 minutes. Digestion of 165 bp PCR product of AGT gene was performed with Tth111I restriction enzyme (Promega, Madison, WI) according to the manufacturer s instructions. The digestion products were analysed on a 3.0% NuSieve GTG (BMA, Rockland, ME) agarose gel prestained with ethidium bromide (0.5 µg/ml) for visualisation under ultraviolet light. In the presence of C at position 704, cleavage by Tth111I generates two fragments of 141 bp and 24 bp. In the presence of T allele at position 704 PCR product undigested and visualised as 165 bp. Analysis of AT 1 R Genotype The AT 1 R A1166C polymorphism was analysed according to Bonnardeaux et al. 10 A 100 ng genomic DNA was amplified by PCR (GeneAmp PCR 9700, PE Applied Biosystems) in a 25 µl mixture of 1x GeneAmp PCR Buffer II (15 mmol/l Tris-HCl, ph 8.0, 50 mmol/l KCl; PE Applied Biosystems Foster City, CA ), 200 µm each datp, dgtp, dctp and dttp (Promega, Madison, WI), 10 pmol sense and antisense primers, 0.2 µl AmpliTaq DNA Polymerase; Applied Biosystems 5 U/µL) and completed with nuclease free water. The sequences of the sense and antisense primers were 5 -GAAGCCTGCACCATGTTTTG-3 and 5 -GATCTGCAACTTGACGACTA-3 (QIAGEN Operon GmbH, Cologne Germany) which yield PCR products of 560 bp. Thermal cycling was: 94 o C for 10 minutes, then thirty-two cycles of 94 o C for 45 seconds, annealing at 56 o C for 50 seconds and extention at 72 o C for 90 seconds, followed by 7 minutes of final extention at 72 o C. The PCR products for AT 1 R gene were analysed on a 2.0% agarose gel (Sigma-Aldrich GmbH, Steinheim, Germany) prestained with ethidium bromide (0.5 µg/ml) for visualisation under ultraviolet light. Gels (15x15 cm) were run at 90 mv in 1xTBE buffer (Sigma-Aldrich Co. St.Louis MO) for 90 minutes. Digestion with DdeI restriction enzyme (New England Biolabs, Beverly, MA) was performed according to the manufacturer s instructions. If the A/C transversion is present at position 1166 nucleotide, the digestion of the PCR product results in two fragments of 444 bp and 116 bp in length, respectively. The amplification products were resolved by electrophoresis on a 2.0% agarose gel and visualised under UV light after staining with ethidium bromide. Statistical Analysis The allele distribution of ACE gene I/D polymorphism, AT 1 R gene A/C polymorphism and AGT gene M/T polymorphism were tested with Hardy-Weinberg equilibrium in patient and control groups. The student t-test was used for comparison of numeric variables within groups and Pearson correlation test was used for correlation of numeric variables. The chi-square test and Fisher s exact test (when appropriate) were used for the comparison of groups. The data was expressed mean±standard deviation (SD) and p values less than 0.05 and odds ratios with 95% confidence intervals not including one were considered as statistically significant. Results Patient s Characteristics There were consanguinous marriages in 24 patients (15.2%) and family history of NS in 15 (9.5%) patients. On admission haematuria and 140

4 hypertension were found in 22 (13.9%) and 15 (9.5%) patients, respectively. Renal dysfunction was found in 19 (12%) and end stage renal disease in 13 (8.2%) of FSGS patients. The diagnosis of MCD was present in 51 (51%) and FSGS in 49 (49%) out of 100 renal biopsy performed patients. The patients without renal biopsy or aged between 2 8 years having selective proteinuria with hypertension, haematuria decreased C3 levels with normal steroid response Table 1 Demographic, clinic, laboratory characteristics and treatment responses of patients. Patient s Characteristics Sex (Male/Female) 93/65 Family history (Present/absent) 15/143 Relationship (Present/absent) 24/134 Hematuria (Present/absent) 22/136 Hypertension (Present/absent) 15/143 FSGS/non-FSGS 49/109 Steroid response (Present/absent) 102/56 Renal dysfunction (Present/absent) 19/139 ESRD (Present/absent) 13/145 Key: FSGS = Focal Segmental Glomerulosclerosis; ESRD = End-stage Renal Disease Table 2 ACE I/D genotype distribution and D allele frequency in patients and controls. n ACE I/D genotype distribution and normal renal functions were included in MCD group. However, at the beginning 102 (64.6%) of the patients were evaluated as steroid sensitive, 31 (19.6%) of them had the diagnosis of steroid dependent in follow-up period. The characteristics of patients are summarised in Table 1. RAS gene Polymorphisms Disease Prevalence Frequencies of II, ID and DD genotype of ACE gene were found to be 12.7%, 41.8%, 45.5% in the patient group and 22.3%, 43.2%, 34.5% in the control group, respectively. AA, AC and CC genotype frequencies of AT 1 R gene were found to be 60.1%, 36.7%, 3.2% in the patient group and 61.5%, 33.8%, 4.6% in the control group, respectively. MM, MT and TT genotype frequencies of AGT gene were 21.5%, 66.5%, 12% in the patient group and 26.7%, 55%, 18.3% in the control group, respectively. There were no statistically significant differences for the C allele of AT 1 R and the T allele of AGT genes between the children with nephrotic syndrome and the control group, but there was statistical significance for D allele of ACE gene (Table 2-4). Corticosteroid Response There was no statistically significant difference for ACE and AT 1 R genotypes between the groups of steroid dependent and steroid responsive patients (p>0.05). There was however a statistically significant difference for AGT TT genotype in the steroid resistant group compared to sensitive controls (OR=4.837, 95% CI= , p=0.01) (Figure 1). Groups II ID DD D allele frequency n (%) n (%) n (%) (%) NS (n=158) 20 (12.7) 66 (41.8) 72 (45.5) 66.4* Controls (n=287) 64 (22.3) 124 (43.2) 99 (34.5) 56.0* *p=0.003 Table 3 AT1R A/C genotype distribution and C allele frequency in patients and controls. AT 1R A/C genotype distribution Peptidergic s Groups AA AC CC C allele frequency n (%) n (%) n (%) (%) NS (n=158) 95 (60.1) 58 (36.7) 5 (3.2) 19.9* Controls (n=287) 176 (61.5) 97 (33.8) 14 (4.6) 21.7* *p=

5 Table 4 AGT M/T genotype distribution and T allele frequency in patients and controls. AGT M/T genotype distribution Groups MM MT TT T allele frequency n (%) n (%) n (%) (%) NS (n=158) 34 (21.5) 105 (66.5) 19 (12) 45.2* Controls (n=287) 77 (26.7) 157 (55) 53 (18.3) 45.8* *p=0.97 Peptidergic s Histological Effects When the histological evaluation was made, we did not find any statistically significant difference between patients with MCD and FSGS and patients with and without FSGS for ACE gene I/D polymorphism, AT 1 R gene A1199C and AGT gene M235T genotypes (p>0.05). Renal Dysfunction There was no statistically significant difference for the ACE gene I/D genotype AT 1 R gene A1199C genotype and for renal dysfunction between two groups (p>0.05). A significantly increased risk in renal dysfunction has been found for the AGT gene TT genotype between the groups (OR=3.189, 95% CI= , p=0.041) (Figure 2). End-stage Renal Disease No difference was found in the distribution of the ACE I/D genotype and AT 1 R A1199C genotype frequencies between groups with respect to the progression to ESRD (p>0.05). When looking at the risk of development ESRD for the AGT TT genotype, there was a statistically significant difference between patients and the control groups (OR=3.852, 95% CI= , p=0.03). In patients with renal dysfunction and ESRD no statistically significant difference was found for the MM or MT genotypes (p>0.05) (Figure 3). Discussion This is the first study in which the effects of all three RAS genes on the clinical progress of childhood primary idiopathic NS patients were studied. In recent years; it was understood that RAS has important effects on endocrine, regulation of blood pressure, fluid-electrolyte and acid-base balance so that it plays a central role on cardiovascular and renal physiopathology. It has been established that Ang II was found at 1,000 times higher concentrations in tubulointerstitial fluid in the nephron than in the systemic circulation. 23 It was also shown that local RAS in the glomerular area, mainly on efferent arterioles, plays the main role in regulating glomerular filtration by changing the effective filtration area. It is also thought that the RAS in the tubulointerstitial area could induce absorption of Na + and HCO 3 through the luminal Na/HCO 3 exchanger, basolateral Na/ HCO 3 co-transporter and Na + /K + ATP ase in the proximal tubules. 24 Percent of patients Percent of patients Steroid responsed NS Steroid resistant NS Renal failure No renal failure p=0.04 II ID DD AA AC CC MM MT TT ACE AT 1R AGT RAS Gene Polymorphisms p=0.01 II ID DD AA AC CC MM MT TT ACE AT 1R AGT RAS Gene Polymorphisms Figure 1 Effects of RAS gene polymorphism on steroid response. Figure 2 Effects of RAS gene polymorphism on renal dysfunction. Ang II has two main receptors, AT 1 and AT 2. Most of the known biological effects occur by activation of AT 1 receptors, for instance vasoconstriction, induction of synthesis and secretion of aldosterone, enhancement of smooth muscle proliferation and hypertrophy, profibrenogenic effects, and secretion of pro-inflammatory cytokines and free oxygen radicals. Induction of AT 2 receptors has the opposite effects. 25 Recently it has been shown that 142

6 Peptidergic s NF- Kappa B has a key role in the action of AT 1 and AT 2 receptors and in particular on Ang II mediated inflammatory events. 26 The good responses in many experimental and clinical studies with ACE inhibitors and/or angiotensin receptor blockers in glomerular and non-glomerular renal diseases support this finding In our study, while we could not find any statistically significant difference in the distribution of polymorphisms in the M/T alleles of the AT 1 R gene and A/C alleles of AGT genes between patients and control groups, we found significant difference in the distribution of I/D alleles of the ACE gene in the NS group as compared to the control group. No significant correlation was detected in the studies of Frishberg et al. with children and Luther et al. with adult NS patients for the frequency of RAS gene polymorphisms. 4,7 In addition no correlation was found in many studies of ACE I/D polymorphism in children with nephrotic syndrome. 8,35,36 In our study, there were no associations between I/D of ACE and A266C gene polymorphism of AT 1 R and response to treatment, histological type and progression to ESRD. Different results have been reported from the studies of ACE I/D polymorphism in children with NS and other renal diseases. 4,7,8,27,28,35-38 In these studies, it was reported that the D polymorphism of ACE gene is a risk factor for the progression to ESRD in patients with FSGS, IgA nephropathy, hypertension and diabetic nephropathy. But it was also reported that no association was detected between the polymorphism of both AT 1 R and AGT and disease progression. It was also noted that the authors were not able to find any correlation due to the limited numbers of the cases in these studies. In this study, for the first time in the literature, we related the effects of RAS gene polymorphisms to renal histology in primary idiopathic NS patients. We detected no significant effect of polymorphisms of any of the three genes on NS histology when we distribute the patients as FSGS and non-fsgs according to the biopsy and steroid response. Although many factors such as environment, genetic and the treatment modality used are clinically important for the prognosis of the disease, the most important one is the response or resistance to the steroid treatment. When we evaluated our patients from this point of view, we found that the TT polymorphism of AGT gene in the steroid resistant group was significantly higher. No significant difference was found for the ACE and AT 1 R gene polymorphism. Many factors were thought to be responsible related to progressive loss of renal function and progression to end-stage renal failure. One of them is the glomerular hyperfiltration. When the renal mass begins to decrease, residual nephrons become hypertrophied in compensation and there Percent of patients No ESRD ESRD p=0.03 II ID DD AA AC CC MM MT TT ACE AT 1R AGT RAS Gene Polymorphisms Figure 3 Effects of RAS gene polymorphism on progression to end-stage renal disease. is progression to glomerular sclerosis as a result of hyperfiltration and intraglomerular hypertension. 39,40 It was shown that, whatever the primary renal disease might be, nephrotic range long-term proteinuria progresses glomeruli to glomerular sclerosis. 41 When the kidney loses nephrons for any reason Ang II-mediated effects are observed: 1. changes in haemodynamics and tubular transport, 2. glomerular and tubular growth by proliferation and hypertrophy, 3. stimulation of fibrogenesis, 4. the infiltration of mononuclear cells leading to glomerulosclerosis and tubulointerstitial fibrosis. As a result renal function decreases rapidly and ESRD becomes inevitable. 24 When we assessed our patients according to RFD and ESRD, we did not observe any effects of ACE and AT 1 R genotypes on the prognosis, but we detected that that the TT polymorphism of AGT was a significant risk factor for progressive RFD and ESRD. We believe that detection of the adverse effects of TT polymorphism of AGT on steroid response may be a significant contribution to understanding mechanisms of progression to ESRD. NS is one of the most commonly seen chronic glomerulopathies in childhood. The histology of FSGS, and especially steroid resistance seem to be the most important factors determining prognosis and accelerating progression to ESRD. On the other hand, the RAS is only one of the factors that effects progression. 42 Conclusions In our study our results have demonstrated that homozygous TT polymorphism of AGT is a genetic risk factor for progression of disease in childhood NS. The observations about the role of the RAS in kidney diseases and the effects of ACE-Is and ARBs, show that RAS blockage can be a treatment modality that will break the pathophysiologic cascade in renal diseases and prevent progression of damage. Larger studies are needed to confirm these findings. 143

7 Peptidergic s References 1. Clark AG, Barrat TM. Steroid-responsive nephrotic syndrome. In: Barrat TM, Avner ED, Harmon WE (eds) Pediatric nephrology. Lipincott Williams and Wilkins, Baltimore, 1999; pp Cortes L, Tejani A. Dilemma of focal segmental glomerular sclerosis. Kidney Int 1996;53:S57-S Boyer M, Meyrier A, Niaudet P, Habib R. Minimal changes and focal segmental glomerular sclerosis. Eds: Davison AM, Cameron JS, Grünfeld JP, Kerr DNS, Ritz E and Winerals CG. Oxford Textbook of Clinical Nephrology, Oxford University Press, Luther Y, Bantis C, Ivens K et al. Effects of the genetic polymorphisms of the renin-angiotensin system on focal segmental glomerulosclerosis. Kidney Blood Press Res 2003;26: Mezzano SA, Ruiz-Ortega M, Egido J. Angiotensin II and renal fibrosis. Hypertension 2001;38: Egido J. Vasoactive hormones and renal sclerosis. Kidney Int 1996;49: Frishberg Y, Becker-Cohen R, Halle D et al. Genetic polymorphisms of the renin-angiotensin system and the outcome of focal segmental glomerulosclerosis in children. Kidney International 1998;54: Oktem F, Sirin A, Bilge I, Emre S, Agachan B, Ispir T. ACE I/D gene polymorphism in primary FSGS and steroid-sensitive nephrotic syndrome. Pediatr Nephrol 2004;19: Rigat B, Hubert C, Alhenc-Gelas F, Rodby R, Gunnels JC, Soubrier F. An insertion/deletion polymorphism in the angiotensin I converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990;86: Bonnardeaux A, Davies E, Jeunemaitre X et al. Angiotensin II type 1 receptor gene polymorphisms in human essential hypertension. Hypertension 1994;24: Jeunemaitre X, Soubrier F, Koteletsev YV et al. Molecular basis of human hypertension: Role of angiotensinogen. Cell 1992;71: Schmidt S, Sharma AM, Zilch O et al. Association of M235T variant of the angiotensinogen gene with familial hypertension of early onset. Nephrol Dial Transplant 1995;10: Morise T, Takeuchi Y, Takeda R. Rapid detection and prevalence of the variants of the angiotensinogen gene in patients with essential hypertension. J Intern Med 1995;237: Caulfield M, Lavender P, Farral M et al. Linkage of the angiotensinogen gene to essential hypertension. N Engl J Med 1994;330: Hingorani AD, Sharma P, Jia H, Hopper R, Brown MJ. Blood pressure and the M235T polymorphism of the angiotensinogen gene. Hypertension 1996;28: Fogarty DG, Harron JC, Hughes AE, Doherty CC, Maxwell AP. The association of an angiotensinogen variant with diabetic nephropathy. (abstract) J Am Soc Nephrol 1995;6: Schmidt S, Strojek K, Grzeszczak W, Ganten D, Ritz E. The M235T variant of the angiotensinogen gene and nephropathy in type 2 diabetes. (abstract) J Am Soc Nephrol 1995;6: Mendoza SA, Reznik VM, Griswold WR, Krensky AM, Yorgin PD, Tune BM. Treatment of steroid-resistant focal segmental glomerulosclerosis with pulse methylprednisolone and alkylating agents. Pediatr Nephrol 1990;4: International study of kidney disease in children. Nephrotic syndrome in children: prediction of histopathology from clinical and laboratory characteristics at the time of diagnosis. Kidney International 1978;13: Update on the 1987 task force report on high blood pressure in children and adolescents: a working group report from the national high blood pressure education program. Pediatrics 1996;98: Schwartz GJ, Brion LP, Spitzer A. The use of plasma creatinine concentration for estimating glomerular filtration rate in infants, children and adolescents. Pediatr Clin North Am 1987;34: Russ AP, Maerz W, Ruzicka V, Stein U, Gross W. Rapid detection of the hypertension-associated Met235Thr allele of the human angiotensinogen gene. Hum Mol Genet 1993;2(5): Seikaly MG, Arant BS, Seney FD. Endogenous angiotensin concentrations in specific intrarenal fluid compartments in the rat. J Clin Invest 1990;86: Wolf G, Bytzmann U, Wenzel UO. The renin-angiotensin system and progression of renal disease: from hemodynamics to cell biology. Nephron Physiol 2003;93: Wiecek A, Chudek J, Kokot F. Role of angiotensin II in the progression of diabetic nephropathy-therapeutic implications. Nephrol Dial Transplant 2003;18(suppl 5): Suzuki Y, Ruiz-Ortega M, Lorenzo O, Ruperez M, Esteban V. Inflammation and angiotensin II. Int J Biochem Cell Biol 2003;35: Lewis EJ, Hunsicker LG, Bain RP, Rohde RD for the Collaborative Study Group. The effects of angiotensinconverting enzyme inhibition of diabetic nephropathy. N Engl J Med 1993;329: Kasiske BL, Kalil RSN, Ma JZ, Liao M, Keane WF. Effects of antihypertensive therapy on the kidney in patients with diabetes: A metaregression analysis. Ann Intern Med 1993;118: Geiger H. Are angiotensin II receptor blockers superior to angiotensin converting enzyme inhibitors with regard to their renoprotective effect? Nephrol Dial Transplant 1997;12: Ichikawa I. Will angiotensin II receptor antagonists be renoprotective in humans. Kidney Int 1996;50: Johnston CL. Angiotensin receptor antagonists: focus on losartan. Lancet 1995;346: Ziai F, Ots M, Provoost AP et al. The angiotensin receptor antagonists, irbesartan, reduces renal injury of experimental chronic renal failure. Kidney Int 1996;50(suppl. 57): Pollock DM, Divish BJ, Polakowski JS, Oppgenorth TJ. Angiotensin II receptor blockade improves renal function in rats with reduced renal mass. J Farmachol Exp Ther 1993;267: Remuzzi A, Perico N, Amuchastegui CS et al. Short- and long-term effect of angiotensin II receptor blockade in rats with experimental diabetes. J Am Soc Nephrol 1993;4: Lee DY, Kim W, Kang SK, Koh GY, Park SK. Angiotensinconverting enzyme gene polymorphism in patients with minimal-change nephrotic syndrome and focal segmental glomerulosclerosis. Nephron 1997;77: Al-Eisa A, Haider MZ, Srivastva BS. converting enzyme gene insertion/deletion polymorphism in idiopathic nephrotic syndrome in Kuwaiti Arab children. Scand J Urol Nephrol 2001;35: Harden PN, Geddes C, Rowe PA et al. Polymorphism in angiotensin converting enzyme gene and progression of IgA nephropathy. Lancet 1995;345: Marre M, Jeunmaitre X, Gallois Y et al. Contribution of genetic polymorphism in the renin-angiotensin system to the development of renal complications in insulin-dependent diabetes. J Clin Invest 1997;99: Hostteter TH, Olson JL, Rennke HG, Venkatachalam VA, Brenner BM. Hiperfiltration in remnant nephrons: A potentially adverse response to renal ablation. Am J Physiol 1981;241: Brenner BM. Nephron adaptation to renal ablation and injury. Am J Physiol 1985;249: Remuzzi G, Ruggenenti P, Benigni A. Pathophysiology of progressive nephropathy. Kidney Int 1997;51: Matsusaka T, Hymes J, Ichikawa I. Angiotensin in progressive renal disease: Theory and practice. J Am Soc Nephrol 1996;7: