사염화탄소가 고혈압흰쥐 신장의 aquaporin과 superoxide dismutase 발현에 미치는 영향

Abstract

간독성인자로 알려진 사염화탄소(CCl4)는 활성산소기를 발생시킴으로 간세포를 손상시키고, 간의 섬유화 또는 간경화를 일으킨다. 또한 간경화의 마지막 단계에서 많은 환자들은 수분조절기전에 장애가 생기며, 이로 인해 복수를 경험하게 된다. 사염화탄소가 대사되면서 발생하는 활성산소기는 신장의 토리(사구체)와 세관의 변성을 유발한다고 알려져 있다. Aquaporin (AQP)은 신장에서 수분흡수를 조절하는 단백질로, 활성산소기는 신장에서 AQP의 발현을 증가시켜 수분조절장애를 일으킨다고 알려져 있다. 활성산소기는 고혈압의 발병에도 관여하며, 항산화효소의 변화에도 영향을 준다고 알려져 있다. 이에 본 연구자는 사염화탄소 처리가 신장의 AQP 발현과 활성산소기를 제거하는 항산화효소 superoxide (SOD)의 발현에 어떤 영향을 주는지 알아보고자 하였으며, 이러한 변화가 고혈압 유무와 그 유병기간에 따라 차이가 나타나는지를 관찰하였다. 8주, 16주 그리고 24주령의 본태성고혈압흰쥐(고혈압흰쥐)와 정상혈압흰쥐를 실험군과 대조군으로 나누었다. 실험군 흰쥐에는 사염화탄소, 대조군 흰쥐에는 올리브오일을 주 2회씩 4주 동안 등에 피하주사(1.6 ml/kg)하였다. 마지막 처치 후 실험동물을 마취시킨 뒤 혈액과 신장조직을 얻었다. 신장의 형태학적 변화는 H&E 염색으로 확인하였고, blood urea nitrogen (BUN), creatinine을 분석하였다. AQP1과 AQP2, CuZnSOD, MnSOD 발현 변화는 면역조직화학염색과 Western blot 분석을 이용하여 관찰하였다. 사염화탄소 처리는 정상혈압군과 고혈압군 모두에서 세관의 종창과 상피세포의 위축 또는 탈락을 유도하였으며, 고혈압흰쥐에서는 사이질조직의 부종과 세관내 cast, 염증세포의 침윤이 관찰되었다. 사염화탄소 처리로 8주령과 16주령 정상혈압흰쥐와 16주령 고혈압흰쥐에서 BUN이 증가되었다. 사염화탄소처리로 creatintine은 감소되는 경향은 있었으나, 8주령 정상혈압군과 고혈압군을 제외하고는 통계적 유의성이 없었다. 대조군에서 관찰된 AQP1은 정상혈압흰쥐와 고혈압흰쥐에서 차이가 없었으며, 주령에 따른 차이도 없었다. 사염화탄소 처리로 8주령과 24주령 고혈압흰쥐의 AQP1은 유의성 있게 증가되었다. 대조군에서 관찰된 AQP2는 정상혈압흰쥐에서 차이가 없었으나, 24주령 고혈압흰쥐에서 관찰된 AQP2는 8주령 고혈압흰쥐보다 유의성 있게 감소되었다. 사염화탄소처리로 AQP2는 8주령과 24주령 고혈압흰쥐에서 유의성 있게 증가되었고, 사염화탄소를 처리한 24주령 고혈압흰쥐의 AQP2는 사염화탄소를 처리한 16주령 고혈압흰쥐보다 유의성 있게 증가되었다. 8주령과 16주령의 대조군에서 관찰된 CuZnSOD와 MnSOD는 정상혈압흰쥐과 고혈압흰쥐에서 차이가 없었으나, 24주령 고혈압흰쥐에서 관찰된 CuZnSOD와 MnSOD는 24주령 정상혈압흰쥐보다 유의성 있게 증가되었다. 사염화탄소 처리는 8주령 고혈압흰쥐에서 CuZnSOD를 유의성 있게 증가시킨 반면, 24주령 고혈압흰쥐의 CuZnSOD와 MnSOD는 사염화탄소를 처리한 8주령과 16주령 고혈압흰쥐보다 유의성 있게 감소시켰다. 이상의 결과를 종합할 때, 사염화탄소에 의한 AQP의 증가와 SOD의 감소는 고혈압흰쥐에서 유의성 있게 나타났으며, 24주령 고혈압흰쥐에서 신장 손상이 크다는 것을 알 수 있었다. 이는 사염화탄소로 유도되는 활성산소기가 유병기간이 긴 고혈압흰쥐에서 AQPs을 증가시켜 수분조절을 변화시킬 것으로 생각되며, 감소된 SODs는 신장손상을 가중시키는데 관여할 것이라 생각할 수 있었다. |Carbon tetrachloride (CCl4) induces hepatocellular damage, resulting in liver cirrhosis by generating reactive oxygen species (ROS). At the stage of decompensated liver cirrhosis, many patients suffer from the abnormal regulation of sodium and water balance such as ascites. Also, the kidney can be directly damaged by CCl4-induced ROS generation. The aquaporin (AQP) is an important transmembrane protein located in the kidney to reabsorb water, and it may be affected by the ROS to alter water balance. Also, ROS is related with the development of hypertension and alteration of antioxidant enzymes. This study was undertaken to investigate the effects of CCl4 on the expression of AQPs (AQP1 and AQP2) and superoxide dismutase (SOD) in the kidney of spontaneously hypertensive rat (SHR) and normotensive Wistar-Kyoto rat (WKY). The SOD is known as a scavenger of ROS, and we hypothesized that oxidative stress in the aged kidneys may be increased by hypertension. Male WKY and SHR were randomly divided into control and CCl4-treated groups at 8, 16 and 24 weeks of age, respectively. The experimental group received olive oil-dissolved CCl4 (1.6 ml/kg) on its back subcutaneous tissue twice a week for 4 weeks, and control animals received vehicle (olive oil) only. After 24 hours following the last injection, blood samples were obtained from abdominal aortas under anesthesia. The kidneys were then removed after sacrificing animals by cervical dislocation. Renal histopathology was examined by H&E stain, and the expression of AQP1, AQP2, CuZn-SOD and Mn-SOD were evaluated by immunohistochemical methods and Western blot analysis. CCl4 treatment induced the tubular swelling, tubular epithelial atrophy or detachment in both WKY and SHR, and interstitial edema, tubular cast and infiltration of leukocyte in SHR. The BUN levels in both WKY and SHR were increased by CCl4 treatment at 16 weeks of age. However serum creatinine levels showed a decreasing tendancy in both WKY and SHR. The expressions of AQP1 showed no difference between control WKY and SHR, but, the expression of AQP1 were increased by CCl4 treatment at 8 and 24 weeks of age SHR. The expressions of AQP2 in 24 week-old control SHR was decreased compared to 8 week-old control SHR. CCl4 treatment increased the expressions of AQP2 at 8 week-old and 24 week-old SHR, and the increasing of AQP2 was more remakable in advanced age. The expressions of CuZnSOD and MnSOD showed no difference between control WKY and SHR at 8 and 16 weeks of age, but were increased at 24 week-old control SHR compared to same aged WKY. Whereas the expression of CuZnSOD was increased by CCl4 treatment in 8 week-old SHR, the expression of both CuZnSOD and MnSOD were decreased by CCl4 treatment in 24 week-old SHR. In summary, CCl4-treated SHR showed an increase in AQP expression and a decrease in SODs at the advanced age. These results suggest that CCl4-induced oxidative stress in the aged hypertensive rats may alter water balance via upregulation of AQPs and accelerate renal damage via downregulation of SODs.; Carbon tetrachloride (CCl4) induces hepatocellular damage, resulting in liver cirrhosis by generating reactive oxygen species (ROS). At the stage of decompensated liver cirrhosis, many patients suffer from the abnormal regulation of sodium and water balance such as ascites. Also, the kidney can be directly damaged by CCl4-induced ROS generation. The aquaporin (AQP) is an important transmembrane protein located in the kidney to reabsorb water, and it may be affected by the ROS to alter water balance. Also, ROS is related with the development of hypertension and alteration of antioxidant enzymes. This study was undertaken to investigate the effects of CCl4 on the expression of AQPs (AQP1 and AQP2) and superoxide dismutase (SOD) in the kidney of spontaneously hypertensive rat (SHR) and normotensive Wistar-Kyoto rat (WKY). The SOD is known as a scavenger of ROS, and we hypothesized that oxidative stress in the aged kidneys may be increased by hypertension. Male WKY and SHR were randomly divided into control and CCl4-treated groups at 8, 16 and 24 weeks of age, respectively. The experimental group received olive oil-dissolved CCl4 (1.6 ml/kg) on its back subcutaneous tissue twice a week for 4 weeks, and control animals received vehicle (olive oil) only. After 24 hours following the last injection, blood samples were obtained from abdominal aortas under anesthesia. The kidneys were then removed after sacrificing animals by cervical dislocation. Renal histopathology was examined by H&E stain, and the expression of AQP1, AQP2, CuZn-SOD and Mn-SOD were evaluated by immunohistochemical methods and Western blot analysis. CCl4 treatment induced the tubular swelling, tubular epithelial atrophy or detachment in both WKY and SHR, and interstitial edema, tubular cast and infiltration of leukocyte in SHR. The BUN levels in both WKY and SHR were increased by CCl4 treatment at 16 weeks of age. However serum creatinine levels showed a decreasing tendancy in both WKY and SHR. The expressions of AQP1 showed no difference between control WKY and SHR, but, the expression of AQP1 were increased by CCl4 treatment at 8 and 24 weeks of age SHR. The expressions of AQP2 in 24 week-old control SHR was decreased compared to 8 week-old control SHR. CCl4 treatment increased the expressions of AQP2 at 8 week-old and 24 week-old SHR, and the increasing of AQP2 was more remakable in advanced age. The expressions of CuZnSOD and MnSOD showed no difference between control WKY and SHR at 8 and 16 weeks of age, but were increased at 24 week-old control SHR compared to same aged WKY. Whereas the expression of CuZnSOD was increased by CCl4 treatment in 8 week-old SHR, the expression of both CuZnSOD and MnSOD were decreased by CCl4 treatment in 24 week-old SHR. In summary, CCl4-treated SHR showed an increase in AQP expression and a decrease in SODs at the advanced age. These results suggest that CCl4-induced oxidative stress in the aged hypertensive rats may alter water balance via upregulation of AQPs and accelerate renal damage via downregulation of SODs.