Nuclear expression of renin-angiotensin system components in NRK-52E renal epithelial cells

Abstract

Introduction Isolated nuclei of sheep proximal tubules express angiotensin 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. Methods RAS components were visualized by immunofluorescent staining in intact cells and protein expression in isolate nuclei. Results An antibody to the Ang I sequence of AGT (AI-AGT) revealed only cytosolic staining, while an antibody to an internal epitope 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/hr that was reduced by aliskiren (0.13 ± 0.01 nmol/mg/hr, n=3, p<0.01); trypsin activation increased activity 3-fold. Peptide staining localized 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. Conclusion 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.

Biochemical evaluation of the renin-angiotensin system: the good, bad, and absolute?

“…Advanced glycation end product exposure significantly reduced ANG-(1-7) and increased metabolism of the peptide that was associated with myofibroblast transition but did not impact ANG II or ANG I levels (9). As these cells express an intracellular RAS, nuclear content of ANG II and ANG-(1-7) was quantified at ϳ60 fmol/mg protein consistent with nuclear immunostaining for both peptides (10).…”

“…The high angiotensin levels compared with tissues likely reflect the lower cellular protein to normalize peptide content, as well as the greater density of angiotensin-expressing cells maintained in culture; however, direct comparisons of angiotensins quantified by RIA (or ELISA) to HPLC-RIA and LC-MS are lacking, and the true cellular content of ANG II or ANG-(1-7) in these cell models is not known. A second issue with cell studies is the presence of circulating RAS components in fetal bovine serum (10). Cells can be maintained in low or serumfree conditions for short periods, but internalized angiotensinogen or renin may persist and contribute to the intracellular expression of angiotensin peptides, particularly if nonrenin proteases that can process the precursor (cathepsins, tonin) are also expressed.…”

“…Danser and colleagues (113,139) suggest that urinary renin may be an alternative measure of an active RAS within the kidney. Quenched fluorescent substrates based on the ANG- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) sequence are available to measure renin. Although this approach does not rely on the detection of ANG I or an exogenous source of angiotensinogen, the sensitivity and specificity of the peptide substrates are not comparable with angiotensinogen.…”

Peptidases and the Renin-Angiotensin System: The Alternative Angiotensin-(1-7) Cascade

“…In the renal NRK-52 epithelial cell line, immunocytochemical staining for renin was evident in the nucleus of these cells [22]. Renin activity as measured by aliskerin-sensitive inhibition of Ang I formation from angiotensinogen was also evident in isolated nuclei in the renal epithelial NRK-52 cells, as well as quantifiable levels of Ang I, Ang II, and Ang-(1-7) [22]. In confirmation of intracellular renin within the kidney, Ishigami and colleagues report a truncated renin transcript expressed in the proximal tubules of the mouse kidney [16].…”

“…In this case, we also quantified the mitochondrial levels of both Ang II and Ang-(1-7) (50-60 fmol/mg protein), which may indicate that the intracellular form of renin contributes to the cellular expression of angiotensin peptides within the kidney [21]. In the renal NRK-52 epithelial cell line, immunocytochemical staining for renin was evident in the nucleus of these cells [22]. Renin activity as measured by aliskerin-sensitive inhibition of Ang I formation from angiotensinogen was also evident in isolated nuclei in the renal epithelial NRK-52 cells, as well as quantifiable levels of Ang I, Ang II, and Ang-(1-7) [22].…”