Renal Expression of Ammonia Transporters in Rats with Amiloride-Induced Renal Tubular Acidosis

아밀로라이드 유발 신세뇨관산증 흰쥐 신장에서 암모니아 운반체의 발현

  • Kim, Seung-Jung (Department of Internal Medicine, Medical Research Institute, Chungbuk National University College of Medicine) ;
  • Kim, Hye-Young (Department of Internal Medicine, Medical Research Institute, Chungbuk National University College of Medicine) ;
  • Choi, Jae-Hyun (Department of Internal Medicine, Medical Research Institute, Chungbuk National University College of Medicine) ;
  • Kim, Jung-Tae (Department of Internal Medicine, Medical Research Institute, Chungbuk National University College of Medicine) ;
  • Kim, Jeong-Eun (Department of Internal Medicine, Medical Research Institute, Chungbuk National University College of Medicine) ;
  • Yeon, Myeong-Ho (Department of Internal Medicine, Medical Research Institute, Chungbuk National University College of Medicine) ;
  • Kim, Sun-Moon (Department of Internal Medicine, Medical Research Institute, Chungbuk National University College of Medicine) ;
  • Kwon, Soon-Kil (Department of Internal Medicine, Medical Research Institute, Chungbuk National University College of Medicine) ;
  • Shin, Kyung-Sub (Department of Laboratory Medicine, Chungbuk National University College of Medicine)
  • 김승중 (충북대학교 의과대학 내과학교실, 의학연구소) ;
  • 김혜영 (충북대학교 의과대학 내과학교실, 의학연구소) ;
  • 최재현 (충북대학교 의과대학 내과학교실, 의학연구소) ;
  • 김정태 (충북대학교 의과대학 내과학교실, 의학연구소) ;
  • 김정은 (충북대학교 의과대학 내과학교실, 의학연구소) ;
  • 연명호 (충북대학교 의과대학 내과학교실, 의학연구소) ;
  • 김선문 (충북대학교 의과대학 내과학교실, 의학연구소) ;
  • 권순길 (충북대학교 의과대학 내과학교실, 의학연구소) ;
  • 신경섭 (충북대학교 의과대학 진단검사의학교실)
  • Published : 2011.06.01

Abstract

Background/Aims: Renal tubular acidosis (RTA) decreases the net acid excretion, predominantly due to a decrease in urinary ammonia excretion. This study examined whether this decrement is associated with changes in the renal expression of the ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), in rats with amiloride-induced RTA. Methods: Male Sprague-Dawley rats were treated intraperitoneally with amiloride (3 mg/kg/day) for 6 days. Rhbg and Rhcg expression was evaluated by immunoblotting and immunohistochemistry. Cell height, total cellular expression, expression in the apical 25% of the cell, and apical expression as a percentage of total expression were quantified using immunohistochemistry with quantitative morphometric analysis. Results: After amiloride treatment for 6 days, the serum bicarbonate level was decreased, and serum potassium was increased. The total urinary ammonia excretion and potassium excretion were decreased. The total Rhbg and Rhcg protein expression levels were not changed in the cortex or outer medulla of the kidney. Light microscopy and immunohistochemistry with quantitative morphometric analysis demonstrated that total Rhcg expression was decreased in the cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) in amiloride-induced RTA, whereas Rhbg immunoreactivity was unchanged. Conclusions: Rats with amiloride-induced RTA have decreased urinary ammonia excretion associated with decreased Rhcg expression in the CCD and OMCD, suggesting that the ammonia transporter Rhcg plays an important role in the pathogenesis of amiloride-induced RTA.

목적: 신세뇨관산증은 요중 산 배설이 감소하는 질환으로, 대부분 요 암모니아 배설의 감소에 의한 것이다. 본 연구는 아밀로라이드로 유발된 신세뇨관산증 흰쥐에서 산 배설의 변화와 암모니아 운반체인 Rh B Glycoprotein (Rhbg)와 Rh C Glycoprotein (Rhcg)의 발현 변화를 확인하여 신세뇨관산증의 병인에 암모니아 운반체의 역할을 규명하고자 하였다. 방법: Sprague-Dawley계 웅성 흰쥐를 사용하였으며, 실험군에는 amiloride (3 mg/kg/day)를 6일간 복강 내 주입하였다. 7일째 대사케이지에 mineral oil을 처리하여 24시간 동안 요를 수집하였다. Rhbg와 Rhcg의 단백 발현을 평가하기 위하여 immunoblot와 면역조직화학염색을 시행하였고, 면역조직 화학염색법을 정량화 분석하였다. 결과: 아밀로라이드 투여군에서 혈중 $tCO_2$ 농도는 대조군 25.6 ${\pm}$ 1.5 mEq/L에 비하여 23.0 ${\pm}$ 1.5 mEq/L로 감소하였으며 (p < 0.05), 혈중 $K^+$농도는 대조군 3.84 ${\pm}$ 0.34 mmol/L에 비하여 4.50 ${\pm}$ 0.46 mmol/L로 약간 높았다(p < 0.05). 아밀로라이드 투여군에서 24시간 요 암모니아 배설은 대조군 0.52 ${\pm}$ 0.07 mmol에 비하여 0.30 ${\pm}$ 0.03 mmol으로 요 암모니아 배설이 감소하였으며(p < 0.05), Urine pH는 양 군 간에 차이가 없어 아밀라이드 투여에 의해 신세뇨관산증이 유발되었음을 확인하였다. 반정량적 immunoblot 검사에서 Rhbg와 Rhcg의 단백 발현은 양 군 간에 유의한 차이가 없었다. 면역조직화학염색과 정량화 분석 결과 아밀로라이드 유발 신세뇨관산증군에서 Rhcg의 면역반응성은 감소하였으나, Rhbg의 면역반응성은 유의한 차이가 없었다. 결론: 아밀로라이드 투여 흰쥐에서 암모니아 배설 감소와 Rhcg의 면역반응성의 감소가 동반되었다. 따라서 아밀로라이드 유발 신세뇨관 산증의 병인에 암모니아 운반체 Rhcg가 중요한 역할을 할 것으로 생각한다.

Keywords

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