Angiotensin I Converting Enzyme 유전자다형성과 IgA 신변증의 진행성 위험인자와의 상관관계

정수환(Shou Huan Zheng),김연수(Yon Su Kim),안규리(Cu Rie Ahn),한진석(Jin Suk Han),김성권(Suhng Gwon Kim),이정상(Jung Sang Lee),채동완(Dong Wan Chae),윤형진(Hyung Jin Yoon),오윤규(Yoon Kyu Oh),진호준(Ho Jun Chin),김현리(Hyun Lee Kim) 대한신장학회 2001 Kidney Research and Clinical Practice Vol.20 No.4

IgA nephropathy(IgAN) is the most common glomerulonephritis(GN) in worldwide, and accounts for 2096 to 40M of all patients with primary GN in Korea. IgAN has diverse clinical courses, but the risk factors affecting the deterioration of renal function are not established. Recently, there were some suggestions that systemic or local expression of peptides of angiotensin system exerts several effects on the progression of renal disease, and the genetic polymorphisms may associated with peptide expression. To evaluate the role of genetic polymorphism of angiotensin I converting enzyme(ACE) polymorphism in the progression of IgAN, the genotypic distributions in 278 biopsy-proven cases of IgAN were studied, which had undergone a renal biopsy at Seoul National University Hospital, between 1979 and 2000. We also compared the genotypes with clinical manifestations to evaluate the clinical implications of genetic polymorphism. The study shows that there was no difference in the ACE genotype frequencies between the patients (II: 26.6%, ID: 55.0%, DD: 18.4%) and normal controls(II: 31.4%, ID: 57.4%, and DD: 11.2%). Seventy- two percent and 48% of patients maintained renal function for 10 years and 20 years after the initial diagnosis in 278 patients, respectively. However, in 153 patients who were followed more than 5 years, the DD genotype was more prevalent in patients with deteriorating renal function than in those with stable renal function(31.8% vs. 13.8%, p=0.0146). Presence of systemic hypertension increased the risk of renal disease progression(OR=3.3), and it was showed 7.4 fold risk whenever the creatinine was increased by 1 mg/dL. Renal disease progression is not associated with DD genotype among normotensive patients at the biopsy. But, in patients with hypertension, II and DD/ID genotypes have an in- creased risk for disease progression when compared with II genotype of normotensive patients(OR=1.4, OR=7.8; respectively). ACE polymorphisms did not have any interaction with the levels of serum creatinine at the time of biopsy in our patients. Our results suggested that ACE genotypes(D allele) affected the progression of IgAN, especially in hypertensive patients. One of the prospects of the present study is the potential for screening high risk individuals, thus helping to develop a practical application of the molecular findings in clinical practice.

IgA 신병증에서 신조직내 Cytokine 및 Chemokine 의 발현과 임상 및 병리학적 소견의 상관성

임춘수(Chun Soo Lim),정수환(Shou Huan Zheng),김연수(Yon Su Kim),안규리(Cu Rie Ahn),한진석(Jin Suk Han),김성권(Suhng Gwon Kim),이정상(Jung Sang Lee),이현순(Hyun Soon Lee),채동완(Dong Wan Chae) 대한신장학회 2001 Kidney Research and Clinical Practice Vol.20 No.4

IgA nephropathy is one of the most common forms of primary glomerulonephritis in adults, and the pathogenetic mechanisms seem to be diverse. Proinflammatory cytokines, Thl/Th2 cytokines, and chemokines would be involved in the pathogenetic pathways and would affect the functional and his- tologic consequences. To evaluate this hypothesis, we tried to quantify the magnitude of intrarenal gene expression of various cytokines(TNF- α, IL-1β, IL- 6, IL-15, IFN- r, IL-2, IL-10) and chemokines(IL-8, RANTES) in 61 renal core biopsy specimens con- firmed as IgA nephropathy by immunofluorescent microscopy. Semiquantitative reverse-transcriptase polymerase chain reactions(RT-PCR) using the internal competitors were done for the quantification of gene transcripts. And using the immunohistochemistry (IHC), we tried to determine the degree of expression and the location of various cytokines and chemokines in renal tissues in 29 patients among the above patients. The IFN- r /IL-10 ratio was higher in patients with renal dysfunction than that in patients with normal renal function(p=0.0483). Gene transcript levels of proinflammatory cytokines(TNF- α, IL-1 β ) were high in patients with significant proteinuria. In patients with severe glomerular sclerosis, the ratio of IFN- z /IL-10 gene transcripts was high(p=0.0363). IL-10 gene transcript level was related to the se- verity of tubulointerstitial damage. The levels of gene expression of TNF- α(p=0.0026), IL-10(p=0.0092) and IFN- r (p=0.0188) were related to the degree of mesangial matrix expansion, and the extent of intrarenal arteriolar lesions correlated with the expression of the IL-8 gene transcript(r=03828, p=0.0033). The cellular infiltration in glomeruli was related with chemokine(IL-8) gene expression, but the relation was not significant statistically. The degree of IgA deposition in glomeruli was related with the expression of IL-6 and IL-15. The expression of intrarenal gene transcripts of various cytokines and chemokines were closely interrelated. Thl or Th2 cytokine polarization was not present in IgA nephropathy. In IHC, TNF- α, IFN- r and IL-2 were immunostained dominantly in mesangial region, but not in tubulointerstitial region. In contrast, positive reactions for IL-10 were observed mainly in tubules. The significant reactions for IL-8 were noted in the periarteriolar and arteriolar areas. The results of RT- PCR and IHC showed positive relationships, but those were not significant statistically. This study suggests that proinflammatory, Thl/ Th2 cytokines and chemokines are involved in the specific processes of inflammation and immunologic injury, and their predominance and the level of expression could determine the pathogenetic processes and the severity of the clinical manifestations in IgA nephropathy.

증식성 및 비증식성 사구체신염에서 Cytokine 과 Chemokine 유전자의 발현 양상

이서진(Seo Jin Lee),김강석(Kang Suk Kim),정수환(Shou Huan Zheng),임춘수(Chun Soo Lim),윤형진(Hyung Jin Yoon),김연수(Yon Su Kim),안규리(Cu Rie Ahn),한진석(Jin Suk Han),노정우(Jung Woo Noh),채동완(Dong Wan Chae),이정상(Jung Sang Lee),김 대한신장학회 2001 Kidney Research and Clinical Practice Vol.20 No.4

Backgroud : Intraglomerular cellular proliferation is one of the major determinants for dividing various glomerulonephritis(GN) into two groups, such as proliferative versus nonproliferative. We hypothesized that this morphological difference could be based on the differential expression of various cytokines and chemokines. To elucidate this hypothesis we quantified the intrarenal gene expression of various cytokines and chemokines, and correlated it with clinical and histological parameters. Methods: Total RNA was extracted from 54 proliferative GN(PGN) core biopsy specimens and 42 nonproliferative GN(NPGN) specirnens. Using the internal competitors RT-PCR was instituted to quantify mRNAs. Results: The magnitude of the gene expressions of IL-2, IFN- r, and IFN- r /IL-10 ratio were signi- ficantly higher in PGN. RANTES and IL-8 had more abundant gene messages in PGN than in NPGN. It was shown that Thl cytokine was upregulated if GN was mediated by immune complexes regardless of cellular proliferation. Upregulation of the IFN- r / IL-10 ratio and TNF- αwas associated with renal dysfunction at the time of renal biopsy. Conclusion Thl, proinflammatory cytokines, and chemokines were more abundant in proliferative GN, and correlated with unfavorable clinical and histologic parameters. We propose that the clinical manifestations and diverse histologic features of human GN are associated with differential expressions of specific cytokines and chemokines. A new way of blocking the actions of these cytokines should be instituted for the treatment and prevention of the progression of GN.

Uteroglobin Exon 1 의 5` Untranslated Region ( UTR ) 의 유전자 다형성이 IgA 신병증의 진행에 미치는 영향

오윤규(Yoon Kyu Oh),권도윤(Do Yoon Kwon),정수환(Shou Huan Zheng),오국환(Kook Hwan Oh),김현리(Hyun Lee Kim),김강석(Kang Seock Kim),김연수(Yon Su Kim),안규리(Cu Rie Ahn),한진석(Jin Suk Han),김성권(Suhng Gwon Kim),이정상(Jung Sang Lee) 대한신장학회 2001 Kidney Research and Clinical Practice Vol.20 No.1

Uteroglobin(UG)은 항염증작용과 면역조절작용을 갖는 단백질로써 인체의 여러 상피세포에서 분비되는 물질이다. Zheng 등은 UG를 없엔 유전자 조작 생쥐에서 인체의 IgA 신병증과 유사한 사구체신염이 발생함을 보고하였다(Nature Med. 1999, 5:1018). UG 유전자의 exon 1의 5' untranslated region(UTR)은 다양한 전사인자(transcriptional factor)에 대한 결합부위를 가지고 있어 유전자 다형성이 UG의 발현에 이상을 가져올 수 있는 가능성을 내포하고 있다. 연자들은 Ugexon 1의 5' UTR부위 중 38번째 핵산의 유전자 다형성(adenine(A) 또는 guanine(G))이 단백질 발현에 영향을 미쳐 IgA 신병증의 진행에 관여할 것이라는 가정하에 본 연구를 수행하였다. 서울대학교병원에서 IgA신병증으로 진단받고 5년 이상 경과된 환자 60명을 대상으로 말초혈액에서 DNA를 추출하여 single strand conformational polymorphism(SSCP), DNA sequencing, 중합연쇄반응(PCR)을 시행한 후 제한효소처리를 시행하여 유전자 다형성을 파악하였으며, 그 결과를 신질환의 진행여부, 혈청내 IgA 신병증으로 진단받고 5년 이상 경과된 환자 60명을 대상으로 말초혈액에서 DNA를 추출하여 single strand conformational polymorphism(SSCP), DNA sequencing, 중합연쇄반응(PCR)을 시행한 후 제한효소처리를 시행하여 유전자 다형성을 파악하였으며, 그 결과를 신질환의 진행여부, 혈청내 IgA-fibronectin(FN) 복합체의 양 등과 비교하였다. 60명의 대조군에서 일반인의 유전자 다형성 빈도를 조사하여 환자들의 빈도와 비교하였다. IgA 신병증 환자의 경우 5' UTR의 38번째 핵산이 AA인 경우가 17명 (28%), AG인 경우가 26명(43%)이었으며 GG인 경우가 17명(28%)으로 정상재조군의 13명(22%), 36명(60%), 11명(18%)과 비교하여 유전자 다형성 빈도는 차이가 없었다. 60명의 IgA 신병증 환자를 86.5개월(60-170; median, range)간 추적관찰 하였을 때 혈청 creatinine(Cr)이 2.0mg/ dL 이상으로 증가하거나 기저값의 2배 이상으로 증가된 경우는 19명이었다. 이를 유전자 아형으로 구분하여 보면 AA형 17명 중 8명에서, AG형 26명 중 10명에서, GG형 17명 중 1명에서 신질환의 진행이 관찰되어 AA형일 경우 신질환의 진행 위험성이 GG형에 비하여 7.6배였으며(p=0.0307) AG형일경우 GG형에 비해 5.4배의(p=0.06) 위험성을 가지고 있었다. UG 유전자 아형에 따른 단백뇨 정도, 고혈압 여부, 혈청 IgA 농도, IgA-FN 복합체 농도에는 차이가 없었다. 조직 검사시 혈청 Cr값은 AA형 1.5mg/dL, AG형 1.3mg/dL, GG형 1.0mg/dL로 AA, AG형이 GG형에 비해 유의하게 높았다(p=0.01,p=0.03). 신질환이 진행한 환자와 정상 신기능을 가진 환자 사이에 나이와 추적관찰기간은 차이가 없었다. 이상의 결과로 연자들은 IgA 신병증의 진행에 UG exon 1 5' UTR의 유전자 다형성이 깊이 관계하며 이는 항염증 및 면역억제기능을 가진 UG의 단백질 발현 차이에서 기인하였을 것이라는 가능성을 제시하였다. Uteroglobin(UG) is an anti-inflammatory/immunomodulatory protein secreted by the epithelial cells of vertebrates. Targeted disruption of UG rendered mouse glomerulonephritis resembling IgA nephro- pathy(IgAN). Sequence analysis on exon 1 of UG showed several putative binding sites for transcription factors, and genetic polymorphisms in this site might influence the expression level of UG as a competitive protein. We speculated that the single nucleotide polymorphism at the 38th nucleotide from the transcription initiation site of UG exon 1 would impact the progression of IgAN. PCR-RFLP was instituted to determine the genetic polymorphism in 60 patients with IgAN. Other measures like SSCP and direct sequencing were also adopted for the verification of polymorphic sites. Seventeen patients with IgAN(28%) were homozygous for adenine at position 38(38AA), 26 patients(43%) were heterozyg- ous(38AG), and 17 patients(28%) were homozygous for the polymorphism(38GG), which was similar to the pattern obtained from the 60 normal controls. The amount of daily proteinuria, presence of hyper- tension, the level of IgA, and the amount of IgA-fibronectin(FN) complexes was similar between the genotypes. Serum IgA-FN level did not influence the progression of disease. However, 8 out of 17 patients (47%) with the AA genotype had progressive disease(PD), 10 of 26 patients(38%) with the AG genotype had PD, and only 1 of 17 patients<6%) with GG homozygocity had PD after 94±30.1 months of follow-up(mean±S.D.). The odds ratio for the progression of renal disease in patients with the AA genotype was 14.93(p=0.0355) and in patients with AG genotype was 12.94(p=0.0496) compared with patients have the GG genotype. Moreover, serum creatinine at the time of kidney biopsy was higher in patients with AA and AG genotypes than in patients with the GG genotype(1.5±0.69: 1.3±0.53: 1.0±0.3lmg/dL; AA: AG: GG; p=0.0137 AA vs. GG; p=0.0269 AG vs. GG). Our results suggest that polymorphism at the 5 UTR region of UG exon 1 is an important marker for the progression of IgAN