Abstract
It has been shown that angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism affects the circulating and cellular levels of ACE and may be a risk factor in several renal diseases. We analyzed the association of ACE gene I/D polymorphism with the clinical presentation of minimal change nephrotic syndrome (MCNS) in a Turkish child population. This study consisted of 97 children with MCNS and 144 healthy controls. Genotyping of ACE gene was performed using polymerase chain reaction (PCR). The distributions of ACE genotypes were II in 13%, ID in 49%, and DD in 38% in patient group, and 9%, 49%, and 42% in control group, respectively. The frequency of the D allele was 63% and that of the I allele was 37% in patients. There were no relevant differences in the allele frequencies and genotypes of ACE I/D polymorphism between patients and controls. However, DD genotype was higher in boys in children with MCNS (78.4%. vs. 50.0%, p = 0.004). The frequencies of DD genotype and D allele in boys were 7.25 and 2.56 times higher than II genotype and I allele in the patient group, respectively. We suggest that DD genotype in boys may be one of the risk factors for MCNS.
INTRODUCTION
Nephrotic syndrome (NS) is characterized by renal glomerular injury resulting in heavy proteinuria, hypoproteinemia, and edema. The International Study of Kidney Disease in ChildrenCitation1 found minimal change disease in 76.6% of children with primary NS.
It has been known that the renin–angiotensin–aldosterone system (RAAS) has a key role in renal pathophysiology, and experimental and clinical studies suggest that angiotensin II (AII) modulates glomerular permeability and selectivity.Citation2 It is synthesized via a pathway that involves several precursor peptides and enzymes, some of them regulated by separate genes. One of the major known actions of AII is to stimulate fluid and sodium retention, by directly acting on tubular cells and through stimulating aldosterone release. More recently, other potential actions of AII have been elucidated, such as in vitro promotion of vascular smooth muscle and glomerular mesangial and renal tubular cell growth.Citation3–5 Also, each of the hemodynamic effects of AII, that is, increased systemic and glomerular blood pressure, and its effects on tissue growth are thought to be involved in progressive renal functional loss.Citation6–8 Although several genetic variabilities have been linked to renal diseases, angiotensin-converting enzyme (ACE) gene insertion (I)/deletion (D) polymorphism is the first genetic polymorphism that received great attention with respect to renal diseasesCitation9,10 and the most commonly investigated gene of the RAAS. The ACE gene is located in chromosome 17q23 and is presented as a diallelic polymorphism in intron 16. Alleles are distinguished by the presence (insertion: I) or absence (deletion: D) of a 287 base pair sequence. There are three genotype variants: II, ID, and DD. High ACE activity in DD genotype compared to ID and II genotype has been demonstrated not only in plasma but also in several tissues, including the heart and kidney,Citation11 and DD genotype has been described in association with a malign influence on renal disease. Therefore, it has been suggested that high local AII concentrations as a result of deletion polymorphism of the ACE gene is associated with proteinuria in chronic renal diseases.Citation12
To our knowledge, there are limited studies on the possible role of ACE gene polymorphism in children with minimal change nephrotic syndrome (MCNS). The aim of this study was to investigate possible relationship between the ACE gene I/D polymorphism and clinical and laboratory findings of MCNS.
MATERIALS AND METHODS
Subjects
Ninety-seven children with clinical presentation of MCNS, ranging in age from 2 to 16 years (57 male, 40 female), were investigated in comparison with 144 unrelated healthy controls (77 male, 67 female). The study was approved by the Local Ethics Committee of Gaziantep University, and informed consent form was obtained from all subjects. Patients and controls were Turkish and ethnically homogeneous.
None of the patients had family history of NS. The mean age of patients was 5.83 ± 3.34 years at the time of diagnosis. The mean follow-up period was 6.21 ± 2.36 years.
NS was defined as heavy proteinuria (>40 mg/m2/h or 4+ proteinuria measured by dipstick), hypoalbuminemia (<2.5 g/dL), hypercholesterolemia (>250 mg/dL), and edema. MCNS was diagnosed from the clinical findings. Patients who had onset of nephrosis between 2 and 5 years of age, with normal renal function, absence of hypertension, absence of hematuria on urine microscopy, and complete response to corticosteroids, were presumed to have minimal change disease on clinical grounds.Citation13 Remission was defined as serum albumin within normal limits and normal urinary protein excretion (negative by dipstick). Patients were considered in relapse if heavy proteinuria and a low serum albumin level were present.Citation13 Children with MCNS were treated with corticosteroids for the initial episode or subsequent relapses of disease. None of the patients in these groups were treated with immunosuppressive medications other than corticosteroids. Prednisolone (60 mg/m2/day) was begun for induction of remission and continued for 4 weeks. After remission had been induced, the dose of prednisolone was reduced monthly and given on alternate days. Treatment was completed within 6 months.
A frequent relapse was defined as three or more relapses within 12 months. Excretion of urinary protein (mg/m2/h) and creatinine clearance was calculated from urine collected during the 24 h period.
ACE Gene I/D Polymorphism Analysis
Genomic DNA was extracted from peripheral blood samples by salting out procedure.Citation14 All DNA samples were analyzed by polymerase chain reaction (PCR) for the presence of ACE gene I/D polymorphism. A technician, blind to the study groups, performed the DNA extractions, PCR amplifications, and restriction fragment length polymorphisms (RFLPs). PCR was performed using flanking primers in ACE gene intron 16. The primers were 5′-CTG-GAG-ACC-ACT-CCC-ATC-CTT-TCT-3′ and 5′-GAT-GTG-GCC-ATC–ACA-TTC-GTC-AGA-T-3′. PCR reactions were performed in 50 μL reaction volumes with 50 pmol each primer, 100 μg genomic DNA, 10× buffer (5 μL, Fermentas Inc., Finland), 1.5 mmol/L of MgCl2, 200 μl/L each deoxy nucleotide triphosphate (dNTP), and 2 U Taq DNA polymerase (Fermentas). Amplification was performed as follows: initial denaturation at 95°C for 2 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 2 min, and extension at 72°C for 3 min. PCR amplification yielded either a 490 bp fragment with the I allele or a 190 bp fragment without the I allele (D allele). All PCR products were electrophoresed on 2% agarose gel-containing ethidium bromide and were visualized under ultraviolet fluorescence and documented with a gel documentation system (Vilber-Lourmat, Cedex, France). To assess the reliability of genotyping, all samples were studied twice with 100% concordance.
Statistical Analysis
Allelic and genotypic frequencies were expressed in percentages. A chi-square (χ2) test was used for statistical analysis of allele frequencies and for the distribution of genotype in patients and controls. The odds ratios (OR) for different association models were calculated with 95% confidence interval (CI) and p-values were calculated with Fisher’s exact test. The p-value was considered to be statistically significant if <0.05. Calculations were done by GraphPad InStat, version 3.05 (GraphPad Software Inc., San Diego, CA, USA).
RESULTS
The ACE genotypes were distributed among our patients as follows: II in 13%, ID in 49%, and DD in 38% (). The frequency of D allele was 63% and that of the I allele was 37% in children with MCNS. There were no significant differences in the distributions of D and I alleles and ACE I/D genotypes between patients and controls (p > 0.05). However, DD genotype was higher in boys in patient group (78.4% vs. 50.0%, p = 0.004). The frequencies of DD genotype and D allele in boys were 7.25 and 2.56 times higher than II genotype and I allele in the patient group (OR = 7.25, 95% CI = 1.73–30.38; OR = 2.56, 95% CI = 1.40–4.66, respectively). The distribution of ACE genotypes and allele frequencies by gender is shown in .
Table 1. The distribution of ACE genotypes and allele frequencies in children with MCNS and controls.
Table 2. The distribution of ACE genotypes and allele frequencies by gender in children with MCNS and controls.
Totally 35 patients were frequent relapsers. There were no statistically significant correlations between the ACE genotypes/allele frequencies and the frequency of relapses and degree of proteinuria in patients.
DISCUSSION
Both experimental and clinical studies suggest that AII is effective on glomerular permeability.Citation2,3 Although the exact mechanism of the ACE gene polymorphism related to the progression of renal disease remains unclear, several studies have demonstrated a more progressive renal functional decline in patients with DD genotype of ACE gene and chronic renal failure.Citation12,15–17 However, this relationship is controversial in some studies.Citation18,19 It has been suggested that DD genotype can have some impact on the rate of renal function loss, but that the impact of the genotype can be obscured by other risk factors.Citation20 Unfortunately, the contrasting studies are hard to compare, because of the heterogeneity in ethnic background, possible interactions with other genetic or environmental renal risk factors, and mostly small sample size.
Genetic polymorphisms of the RAAS have been mainly investigated in focal segmental glomerulosclerosis (FSGS), one of the most severe renal diseases in children. It has been found that homozygosity for the ACE I allele may have a protective effect in children with FSGS and can serve as a positive prognostic indicator at diagnosis, while the D allele may exert a detrimental dominant effect on outcome.Citation21 Recently, it has also been shown that the DD genotype may be a risk factor for the development of progressive renal impairment in children with FSGS.Citation22 Another study demonstrated that ACE I/D polymorphism does not contribute to the steroid resistance, but carriers of I/D genotype introduce 2.8-fold risk for NS.Citation23
The genetic polymorphism of ACE gene I/D polymorphism in children with idiopathic nephrotic syndrome (INS) as well as its relationship with patients’ clinical response to steroid therapy has been investigated in several studies. Serdaroglu et al.Citation24 found that the D allele frequency was higher in NS patients than healthy controls, and DD or ID genotype was related with frequent relapses, while ACE gene I/D polymorphism was not important in laboratory and histological findings and in the progression of disease in children with NS.
Tsai et al.Citation25 showed that INS was associated with a higher incidence of the DD genotype, especially in nonsteroid-sensitive patients, and suggested that the DD genotype may be a risk factor for INS and play a role in the clinical response to steroids.
In literature, there are few studies about the ACE gene polymorphism in MCNS, the most benign form of childhood glomerulonephritis. Lee et al.Citation26 evaluated the ACE gene I/D polymorphism in 85 NS patients (55 MCNS, 30 FSGS cases) and found that the distribution of ACE genotypes in MCNS was similar to that in controls, while the DD genotype was more frequent in FSGS than in MCNS. Al-Eisa et al.Citation27 investigated the association of ACE gene I/D polymorphism with the clinical presentation of 54 children with INS in Kuwait. They observed that the clinical manifestation of the disease was considerably severe in cases with DD genotypes compared with cases having ID and II genotypes, and 73% of the INS cases with minimal change lesion had a DD genotype.Citation27 Sasongko et al.Citation28 found that the genotype frequencies of ACE gene among steroid-sensitive MCNS patients were consistent with those of the controls, suggesting that there was no relationship between each genotype and steroid sensitivity. More recently, a meta-analysis was performed to evaluate the association between ACE I/D gene polymorphism and MCNS susceptibility. It has been shown that the D allele or DD genotype might be a significant genetic molecular marker for MCNS susceptibility in Asians and overall populations, but not for Caucasians and Africans, and they stressed that more larger and rigorous genetic epidemiological investigations are required to further explore this association.Citation29
Our study demonstrated no influence of the ACE I/D gene polymorphism (genotype distribution and allele frequency) on the frequency of relapses and the degree of proteinuria in clinical MCNS. Considering the benign course of the disease, this result may be expected. Interestingly, we found that DD genotype was higher in boys with MCNS. In the literature, it has been known that there is a male preponderance in children with MCNS, and the incidence of disease in boys is higher than girls (3/2).Citation13 Although the reason of male predominance has not been known, we suggest that DD genotype in boys may be one of the risk factors for MCNS. In spite of the small number of subjects used in this study, the sample was well defined. However, a prospective study in a larger group of patients should be carried out to confirm this hypothesis.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES
- Nephrotic syndrome in children: Prediction of histopathology from clinical and laboratory characteristics at time of diagnosis. A report of the International Study of Kidney Disease in Children. Kidney Int. 1978;13(2):159–165.
- Remuzzi G, Perico N, Macia M, Ruggenenti P. The role of renin-angiotensin aldosterone system in the progression of chronic kidney disease. Kidney Int Suppl. 2005;99:S57–S65.
- Remuzzi G, Ruggenenti P, Benigni A. Understanding the nature of renal disease progression. Kidney Int. 1997;51(1):2–15.
- Wolf G. Molecular mechanisms of angiotensin II in the kidney: Emerging role in the progression of renal disease: Beyond renal hemodynamics. Nephrol Dial Transplant. 1998;13(5):1131–1142.
- Dzau VJ. Cell biology and genetics of angiotensin in cardiovascular disease. J Hypertens Suppl. 1994;12(4):3–10.
- Raij L, Keane WF. Glomerular mesangium: Its function and relationship to angiotensin II. Am J Med. 1985;79(3C):24–30.
- Buschhausen L, Seibold S, Gross O, Matthaeus T, Weber M, Schulze-Lohoff E. Regulation of mesangial cell function by vasodilatory signaling molecules. Cardiovasc Res. 2001;51(3):463–469.
- Siragy HM, Carey RM. Role of the intrarenal renin-angiotensin-aldosterone system in chronic kidney disease. Am J Nephrol. 2010;31(6):541–550.
- Yoshida H, Kon V, Ichikawa I. Polymorphisms of the renin-angiotensin system genes in progressive renal diseases. Kidney Int. 1996;50(3):732–744.
- Kitamura H, Moriyama T, Izumi M, . Angiotensin I-converting enzyme insertion/deletion polymorphism: Potential significance in nephrology. Kidney Int Suppl. 1996;55:S101–S103.
- Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, 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(4):1343–1346.
- Navis G, van der Kleij FG, de Zeeuw D, de Jong PE. Angiotensin-converting enzyme gene I/D polymorphism and renal disease. J Mol Med. 1999;77(11):781–791.
- Nash MA, Edelmann Jr CM, Burnstein J, Barnett HL. Minimal change nephrotic syndrome, diffuse mesangial hypercellularity and focal glomerular sclerosis. In: Edelmann Jr CM, ed. Pediatric Kidney Disease. 2nd ed., Boston, MA: Little Brown; 1992:1267–1290.
- Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16(3):1215.
- Jardine AG, Padmanabhan N, Connell JM. Angiotensin-converting enzyme gene polymorphisms and renal disease. Curr Opin Nephrol Hypertens. 1998;7(3):259–264.
- Hadjadj S, Belloum R, Bouhanick B, . Prognostic value of angiotensin-I converting enzyme I/D polymorphism for nephropathy in type 1 diabetes mellitus: A prospective study. J Am Soc Nephrol. 2001;12(3):541–549.
- Vleming LJ, van der Pijl JW, Lemkes HH, . The DD genotype of the ACE gene polymorphism is associated with progression of diabetic nephropathy to end stage renal failure in IDDM. Clin Nephrol. 1999;51(3):133–140.
- Perna A, Ruggenenti P, Testa A, . ACE genotype and ACE inhibitors induced renoprotection in chronic proteinuric nephropathies. Kidney Int. 2000;57(1):274–281.
- Pei Y, Scholey J, Thai K, Suzuki M, Cattran D. Association of angiotensinogen gene T235 variant with progression of immunoglobulin A nephropathy in Caucasian patients. J Clin Invest. 1997;100(4):814–820.
- Samuelsson O, Attman PO, Larsson R, . Angiotensin I-converting enzyme gene polymorphism in non-diabetic renal disease. Nephrol Dial Transplant. 2000;15(4):481–486.
- Frishberg Y, Becker-Cohen R, Halle D, . Genetic polymorphisms of the renin-angiotensin system and the outcome of focal segmental glomerulosclerosis in children. Kidney Int. 1998;54(6):1843–1849.
- 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;19:384–349.
- Celik US, Noyan A, Bayazit AK, . ACE gene polymorphism in Turkish children with nephrotic syndrome. Ren Fail. 2006;28(5):401–403.
- Serdaroglu E, Mir S, Berdeli A, Aksu N, Bak M. ACE gene insertion/deletion polymorphism in childhood idiopathic nephrotic syndrome. Pediatr Nephrol. 2005;20(12):1738–1743.
- Tsai IJ, Yang YH, Lin YH, Wu VC, Tsau YK, Hsieh FJ. Angiotensin-converting enzyme gene polymorphism in children with idiopathic nephrotic syndrome. Am J Nephrol. 2006;26(2):157–162.
- Lee DY, Kim W, Kang SK, Koh GY, Park SK. Angiotensin-converting enzyme gene polymorphism in patients with minimal-change nephrotic syndrome and focal segmental glomerulosclerosis. Nephron. 1997;77(4):471–473.
- Al-Eisa A, Haider MZ, Srivastva BS. Angiotensin converting enzyme gene insertion/deletion polymorphism in idiopathic nephrotic syndrome in Kuwaiti Arab children. Scand J Urol Nephrol. 2001;35(3):239–242.
- Sasongko TH, Sadewa AH, Kusuma PA, . ACE gene polymorphism in children with nephrotic syndrome in the Indonesian population. Kobe J Med Sci. 2005;51(3–4):41–47.
- Zhou TB, Qin YH, Su LN, Lei FY, Huang WF, Zhao YJ. Relationship between angiotensin-converting enzyme insertion/deletion gene polymorphism and susceptibility of minimal change nephrotic syndrome: A meta-analysis. Int J Nephrol. 2011;2011:360357.