Publication: Deneysel glomerülonefrit modelinde FK506 etkisinin immünohistokimya yöntemiyle araştırılması
Abstract
Anti-Thy-1.1 glomerülonefrit, mezangial hücre proliferasyonu ve mezangial matriks artışı ile karakterize deneysel bir glomerülonefrit modelidir. Bu çalışmada deneysel anti-Thy-1.1 glomerülonefrit modelinde farklı immünohistokimyasal yöntemlerle PCNA ve a-SMA ekspresyonunu araştırdık. İmmünohistokimyasal boyama, protein ekspresyonlarını araştırmak ve ölçmek için yaygın olarak kullanılmaktadır. Double boyama yöntemi parafin kesitler ve sitolojik yaymalara uygulanabilen bir yöntemdir. Bu yöntem özel teçhizat veya teknik donanım gerektirmez. Takrolimus (FK506), otoimmün hastalıkların tedavisinde ve organ nakillerinden sonra kullanılan immünsupressif bir ilaçtır. Bu çalışmanın amacı, deneysel glomerülonefrit modelinde Prolifere Hücre Nükleer Antijen (PCNA) ve a-düz kas aktin a-Smooth Muscle Actin (a-SMA) immünekpresyonlarını, Streptavidin-Biotin / Alkalen Fosfataz kompleks (Str.ABC/ AP), Streptavidin-Biotin/ Horseradish Peroksidaz kompleks (Str.ABC/ HRP) ve double immünohistokimya (Str.ABC/ HRP ve Str.ABC/ AP) boyama metotları ile araştırmaktır. Ayrıca deneysel glomerülonefrit modelinde takrolimus etkisini de gözden geçirmektir. Yirmi Wistar Albino sıçan bu çalışmada 5'erli dört gruba ayrılmıştır. Grup I Kontrol (K): Dört hafta 0,1ml/ 100g serum fizyolojik (SF) intravenöz uygulanmıştır; grup II Glomerülonefrit (GN): 0.gün anti-Thy 1.1 antikor (0,25µg/ 100g) intravenöz uygulanmıştır; grup III Glomerülonefrit+FK506 (GFK): önce anti-Thy 1.1 antikor (0,25µg/ 100g) 0.günde, sonra FK506 (1mg/ kg) iki hafta süresince uygulanmıştır. Grup IV Kontrol+FK506 (KFK): önce iki hafta SF (0,1ml/ 100g), daha sonra iki hafta boyunca FK506 (1mg/ kg ) uygulanmıştır. Çalışma sonunda serumda kreatinin, idrarda kreatin, kreatin klirensi ve proteinüri bakılmıştır. Böbrek dokusunda; ışık mikroskobunda, glomerüler ve tubulointerstisyel alanda, PCNA ve a-SMA immünekspresyonu Str.ABC/ HRP, Str.ABC/ AP ve double immünohistokimya ile boyanan lamlarda değerlendirilmiştir. Çalışma sonunda; kontrol grubuna göre, glomerülonefrit grubunda glomerüler hücre proliferasyonu ve tubulointerstisyel hasar anlamlı ölçüde artmıştır. Tüm gruplarda uyguladığımız immünohistokimyasal metotlar ile a-SMA ekspresyonu birbirine yakın bulunmuştur. a-SMA nin tubulointerstisyal alanda immünekspresyonu Str.ABC/ AP ve Str.ABC/ HRP boyama yöntemlerinde GFK grubunda GN grubuna göre anlamlı derecede azalmıştır. Tüm immünohistokimya yöntemlerinde tubulointerstisyel alan ve glomerüllerde PCNA immünekspresyonu K grubuna göre GN grubunda, KFK grubuna göre GFK'de arttığı bulunmuştur. Sonuçta double immünohistokimya boyama yönteminin, aynı bölgede iki farklı antijenin araştırılmasında kullanılabilecek güvenilir ve basit bir yöntem olduğu gösterilmiştir. EFFECT OF FK506 IN EXPERIMENTAL MESANGIAL PROLIFERATIVE GLOMERULONEPHRITIS BY IMMUNOSTAINING METHODS
The anti-Thy-1.1 model is a rat model of mesangial proliferative glomerulonephritis characterized by mesangial cell proliferation and accumulation of mesangial matrix expansion. In present study we investigate the expression of a-smooth muscle actin (a-SMA) and proliferative cell nuclear antigen (PCNA) in renal cortex in the rat model of mesangial proliferative glomerulonephritis induced with anti-Thy-1.1 antibody by different immunohistochemical methods. Immunostaining is used widely for detection and measurement of protein expression. The double staining method described may have wide application since it can be used both for paraffin sections and cell smears. It does not require specialized technique or aparatus. Tacrolimus (FK506) is an immunsuppressive drug used for the treatment of autoimmune disease and after organ transplantations. The aim of our study to evaluate the immunexpression PCNA and a-SMA in anti-Thy-1.1 diffuse experimental glomerulonephritis rat model by Str.ABC/ HRP, StrABC/ AP and double (Str.ABC/ HRP and Str.ABC/ AP) immunohistochemistry methods. Also we want to investigate tacrolimus effect in this experimental glomerulonephritis model. Twenty male Wistar Albino rats were divided into four groups of fifth: Group I Control (C): 5 treated 0,1ml / 100g normal saline intravenously (IV) for four weeks; group II glomerulonephritis (GN): 5 treated with anti-Thy 1.1 (0,25µg/ 100g) IV at 0 day; group III Glomerulonephritis+FK506 (GFK): 5 treated with Anti-Thy-1.1 (0,25µg/ 100g) IV at 0 day then FK506 (1mg/ kg) for two weeks. Group IV Control+FK506 (CFK): 5 treated with normal saline (0.1mg/ 100mg) both for two weeks then FK506 (1mg/ kg) for two weeks. Serum creatinine, urinary creatinine, creatinine clearance and proteinuria were performed at the end of the study period. Renal tissue were assessed for light microscopic findings glomerul and tubulointerstitial injury and PCNA, a-SMA expression were semiquantatitively scored on glomeruli and tubulointerstitium by Str.ABC/ HRP, Str.ABC/ AP and double immunohistochemical methods staining slides. At the end of the study period morphological changes including tubulointerstitial injury and glomerular cell proliferations were significantly increased in glomeulonephritis group compared to control group. With in immunohistochemical methods, glomerular immunoexpression of a-SMA was similar in all groups. Tubulointestitial immunoexpression of a-SMA was significantly statical decreased in Str.ABC/ AP and Str.ABC/ HRP in GFK compared to GN group. Immunoexpression of PCNA in all immunohistochemical methods on tubulointerstitial and glomeruli was significant increased in GN group compared to C, and GFK group compared to CFK. In conclusion double ımmunostaing was the method to detect two antigen in same site. That method was simple and reliable.
The anti-Thy-1.1 model is a rat model of mesangial proliferative glomerulonephritis characterized by mesangial cell proliferation and accumulation of mesangial matrix expansion. In present study we investigate the expression of a-smooth muscle actin (a-SMA) and proliferative cell nuclear antigen (PCNA) in renal cortex in the rat model of mesangial proliferative glomerulonephritis induced with anti-Thy-1.1 antibody by different immunohistochemical methods. Immunostaining is used widely for detection and measurement of protein expression. The double staining method described may have wide application since it can be used both for paraffin sections and cell smears. It does not require specialized technique or aparatus. Tacrolimus (FK506) is an immunsuppressive drug used for the treatment of autoimmune disease and after organ transplantations. The aim of our study to evaluate the immunexpression PCNA and a-SMA in anti-Thy-1.1 diffuse experimental glomerulonephritis rat model by Str.ABC/ HRP, StrABC/ AP and double (Str.ABC/ HRP and Str.ABC/ AP) immunohistochemistry methods. Also we want to investigate tacrolimus effect in this experimental glomerulonephritis model. Twenty male Wistar Albino rats were divided into four groups of fifth: Group I Control (C): 5 treated 0,1ml / 100g normal saline intravenously (IV) for four weeks; group II glomerulonephritis (GN): 5 treated with anti-Thy 1.1 (0,25µg/ 100g) IV at 0 day; group III Glomerulonephritis+FK506 (GFK): 5 treated with Anti-Thy-1.1 (0,25µg/ 100g) IV at 0 day then FK506 (1mg/ kg) for two weeks. Group IV Control+FK506 (CFK): 5 treated with normal saline (0.1mg/ 100mg) both for two weeks then FK506 (1mg/ kg) for two weeks. Serum creatinine, urinary creatinine, creatinine clearance and proteinuria were performed at the end of the study period. Renal tissue were assessed for light microscopic findings glomerul and tubulointerstitial injury and PCNA, a-SMA expression were semiquantatitively scored on glomeruli and tubulointerstitium by Str.ABC/ HRP, Str.ABC/ AP and double immunohistochemical methods staining slides. At the end of the study period morphological changes including tubulointerstitial injury and glomerular cell proliferations were significantly increased in glomeulonephritis group compared to control group. With in immunohistochemical methods, glomerular immunoexpression of a-SMA was similar in all groups. Tubulointestitial immunoexpression of a-SMA was significantly statical decreased in Str.ABC/ AP and Str.ABC/ HRP in GFK compared to GN group. Immunoexpression of PCNA in all immunohistochemical methods on tubulointerstitial and glomeruli was significant increased in GN group compared to C, and GFK group compared to CFK. In conclusion double ımmunostaing was the method to detect two antigen in same site. That method was simple and reliable.