Nefritisli köpeklerin serum ve idrar protein fraksiyonlarının elektroforetik olarak ayrımı ve idrar proteinlerinin immünoblot tekniği ile incelenmesi
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Date
2002-08-22
Authors
Yalçın, Abdullah
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Publisher
Uludağ Üniversitesi
Abstract
Yirmi beş adedi nefritis şüpheli ve 25 adedi sağlıklı olmak üzere toplam 50 adet köpekte gerçekleştirilen çalışmada nefritisli köpeklerin serum ve idrar protein fraksiyonlarındaki değişimlerin incelenmesi, idrar proteinlerinin immünoblot tekniği ile belirlenmesi amaçlandı. Gruplar arasında idrar toplam protein, kreatinin düzeyleri ve protein/kreatinin oranı p<0.001 düzeyinde önemli bulundu. Serum toplam protein ve albumin düzeyleri ve A/G (albümin/globülin) oranı iki grup arasında p<0.05 düzeyinde önemli bulunurken, globulin ve globulin fraksiyonları düzeylerinde istatistiki açıdan anlamlı bir farklılık saptanmadı. İdrar proteinlerinin SDS-PAGE (sodyum dodesil sülfat poliakrilamit jel elektroforezi)'i sonucu jel üzerinde 98,76, 66, 55, 45, 38, 30, 22, 16 ve 14-10 kd (kilodalton) arası moleküler ağırlığa sahip 10 protein bantı belirlendi Protein bantlarının görülme sıklıkları açısından 98 kd bantında farklılık gözlenmezken; 76, 66, 55, 45, 38, 30, 22, 16 ve 14-10 kd bantlarının görülme sıklıkları iki grup arasında sırasıyla p<0.01, p<0.01, p<0.01, p<0.05, p<0.05, p<0.05, p<0.01, p<0.01 ve p<0.001 düzeyinde istatistiki öneme sahipti. İdrarlarında HMW (yüksek moleküler ağırlıklı)+MMW (orta moleküler ağırlıklı)+LMW (düşük moleküler ağırlıklı) ya da MMW+LMW proteinlere rastlananlar glomerülotübüler proteinürili, sadece LMW proteinlere rastlananlar tübüler proteinürili olarak değerlendirildi. Serum proteinlerinin SDS-PAGE'i sonucunda belirgin olarak 76, 66, 55, 38, 30 ve 26 kd bantları elde edildi. İmmünoblot ile köpeklerin idrarlarında transferrin, aı - mikroglobülin, retinol bağlayıcı protein ve pY mikroglobülin belirlendi Nefritisli köpeklerin % 92'sinde retinol bağlayıcı protein, % 100'ünde ar mikroglobülin ve % 96'sında transferrin pozitif bulundu, {^mikroglobülin sadece bir hastada belirlendi Sonuç olarak, elektroforetik analizler serum protein fraksiyonları iyi bir şekilde belirlenmekle beraber, bu verilerin nefritis veya proteinürileri anlamada diagnostik bir öneminin olmadığı düşünüldü. İdrar proteinleri SDS-PAGE ile iyi bir şekilde ayrıldı ve immünoblot analizi ile proteinürileri ve orijinlerini anlamada yardımcı olabilecek bazı proteinler belirlendi Elde edilen sonuçlar ışığında, protemürinin ve idrar proteinlerindeki değişimlerin nefritisin en önemli bulgularından biri olduğu ve SDS-PAGE ile beraber immünoblotun bu değişimleri anlamada klinik ve deneysel olarak yararlı bir bilgi sağlayacağı kanısına varıldı.
The objective of this study was to investigate the changes of serum and urine protein fractions of dogs with nephritis, and to detect some urine proteins of dogs using immunoblotting. A total of 50 dogs, 25 of them were suspected of nephritis, and 25 of them were healthy, were used in the study. Urine total protein and urine creatinine levels, and urine protein/urine creatinine ratios were found significantly different between groups at the level of pO.001. While statistically significant differences were found between two groups for serum total protein, albumin and A/G (albumin/globulin) ratios; there was no significance between groups for globulin and globulin fractions. We have been able to detect 10 different patterns at the molecular weights of 98 kd (kilo dalton), 76 kd, 66 kd, 55 kd, 45 kd, 38 kd, 30 kd, 22 kd, 16 kd and 14-10 kd on the stained gels by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) of urine proteins. There were statistically significant differences in the visualization frequencies of these patterns except 98 kd between two groups at the levels of p<0.01, p<0.01, pO.Ol, p<0.05, p<0.05, p<0.05, p<0.01, p<0.01 and p<0.001, respectively. While proteinurias which consisted of HMW (high molecular weight)+MMW (middle molecular weight)+LMW (low molecular weight) proteins or MMW+LMW proteins was named as glomerulotubular proteinuria, proteinurias which consisted of LMW proteins only was named as tubular proteinuria. We detected 76 kd, 66 kd, 55 kd, 38 kd, 30 kd ve 26 kd patterns on the gels after separation of serum proteins with SDS-PAGE. We have been able to identify transferrin, ai-microglobulin, retinol binding protein and β2-microglobulin in urine of dogs using immunoblotting. Retinol binding protein, ai- microglobulin and transferrin have been identified in 92%, 100% and 96% of urines of diseased dogs, respectively. p2-microglobulin was detected only in one nephritic sample. In conclusion, although serum proteins were successfully separated electrophoretically, it was thought that these findings had not a diagnostic importance in evaluating nephritis and proteinuria. Urine proteins of dogs were also successfully separated based on molecular weights by using SDS-PAGE and some proteins which may be useful in understanding of proteinuria and the origin of proteinuria have been detected IIIusing immunoblotting in the urine of dogs. Based on the results obtained, it can be said that proteinuria and changes in the urine proteins are still one of the most common findings in nephritis, and, it can also be said that SDS-PAGE together with immunoblotting provides a reliable information which is experimentally and clinically useful in understanding those changes.
The objective of this study was to investigate the changes of serum and urine protein fractions of dogs with nephritis, and to detect some urine proteins of dogs using immunoblotting. A total of 50 dogs, 25 of them were suspected of nephritis, and 25 of them were healthy, were used in the study. Urine total protein and urine creatinine levels, and urine protein/urine creatinine ratios were found significantly different between groups at the level of pO.001. While statistically significant differences were found between two groups for serum total protein, albumin and A/G (albumin/globulin) ratios; there was no significance between groups for globulin and globulin fractions. We have been able to detect 10 different patterns at the molecular weights of 98 kd (kilo dalton), 76 kd, 66 kd, 55 kd, 45 kd, 38 kd, 30 kd, 22 kd, 16 kd and 14-10 kd on the stained gels by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) of urine proteins. There were statistically significant differences in the visualization frequencies of these patterns except 98 kd between two groups at the levels of p<0.01, p<0.01, pO.Ol, p<0.05, p<0.05, p<0.05, p<0.01, p<0.01 and p<0.001, respectively. While proteinurias which consisted of HMW (high molecular weight)+MMW (middle molecular weight)+LMW (low molecular weight) proteins or MMW+LMW proteins was named as glomerulotubular proteinuria, proteinurias which consisted of LMW proteins only was named as tubular proteinuria. We detected 76 kd, 66 kd, 55 kd, 38 kd, 30 kd ve 26 kd patterns on the gels after separation of serum proteins with SDS-PAGE. We have been able to identify transferrin, ai-microglobulin, retinol binding protein and β2-microglobulin in urine of dogs using immunoblotting. Retinol binding protein, ai- microglobulin and transferrin have been identified in 92%, 100% and 96% of urines of diseased dogs, respectively. p2-microglobulin was detected only in one nephritic sample. In conclusion, although serum proteins were successfully separated electrophoretically, it was thought that these findings had not a diagnostic importance in evaluating nephritis and proteinuria. Urine proteins of dogs were also successfully separated based on molecular weights by using SDS-PAGE and some proteins which may be useful in understanding of proteinuria and the origin of proteinuria have been detected IIIusing immunoblotting in the urine of dogs. Based on the results obtained, it can be said that proteinuria and changes in the urine proteins are still one of the most common findings in nephritis, and, it can also be said that SDS-PAGE together with immunoblotting provides a reliable information which is experimentally and clinically useful in understanding those changes.
Description
Keywords
SDS-PAGE, İdrar proteinleri, Nefritis, Köpek, İmmünoblot, Urine proteins, Nephritis, Dog, immunoblotting
Citation
Yalçın, A. (2002). Nefritisli köpeklerin serum ve idrar protein fraksiyonlarının elektroforetik olarak ayrımı ve idrar proteinlerinin immünoblot tekniği ile incelenmesi. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.