Abstract
Isolated nuclei of sheep proximal tubules express angiotensin (Ang) receptors as well as angiotensinogen (AGT) and renin. The present study characterized the NRK-52E tubular epithelial cell line for the intracellular expression of renin-angiotensin system (RAS) components. RAS components were visualized by immunofluorescent staining in intact cells and protein expression in isolated nuclei. An antibody to the angiotensin I (Ang I) sequence of AGT (AI-AGT) revealed only cytosolic staining, while an antibody to an internal sequence of AGT (Int-AGT) revealed primarily nuclear staining. Immunoblots of nuclear and cytosolic fractions confirmed the differential cell staining of AGT. Immunostaining for renin was present on nuclei of intact cells. Nuclear renin activity averaged 0.77±0.05 nmol/mg protein/h that was reduced by aliskiren (0.13±0.01 nmol/mg/h, n=3, p<0.01); trypsin activation increased activity three-fold. Peptide staining localized angiotensin II (Ang II) and Ang-(1-7) to the nucleus and peptide content averaged 59±2 and 57±22 fmol/mg (n=4), respectively. Peptide metabolism in isolated nuclei revealed the processing of Ang I to Ang-(1-7) by thimet oligopeptidase. We conclude that the NRK-52E cells express an intracellular RAS localized to the nucleus and may be an appropriate cell model to elucidate the functional relevance of this system.
Highlights
Isolated nuclei of sheep proximal tubules express angiotensin receptors as well as angiotensinogen (AGT) and renin
We conclude that the NRK-52E cells express an intracellular renin-angiotensin system (RAS) localized to the nucleus and may be an appropriate cell model to elucidate the functional relevance of this system
The Ang I sequence of AGT (AI-AGT) antibody recognized the N terminus or Ang I sequence of rat AGT while the internal epitope of AGT (Int-AGT) antibody was directed against an internal sequence distal to Ang I [17]
Summary
Isolated nuclei of sheep proximal tubules express angiotensin receptors as well as angiotensinogen (AGT) and renin. Originally identified as a classic endocrine system, the evidence clearly reveals a local or tissue RAS in various organs including the kidney, heart, adrenals and brain [1,2,3]. In this regard, an intracellular system localized to cellular organelle including the nucleus and mitochondria have been described in the both tubular epithelial and mesangial cells of the kidney, as well as the myocytes and fibroblasts of the heart [4,5,6,7,8,9,10,11]. The prorenin receptor (PRR) is primarily localized intracellularly rather than on the cell membrane suggesting that the receptor may contribute to a functional intracellular RAS [15]
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