Erythrocyte miRNA 144 and miRNA 451 as Cell Aging Biomarkers in African American Adults

Objective: MicroRNAs (miRNA) are novel critical regulators of cell proliferation and human disease, including diabetes mellitus and cancer. The aim of this study was to evaluate the expression of circulating erythrocytes (E) miRNA-144 and miRNA-451 expression in African Americans Adults (AAA) as a biomarker of cell aging. Methods: The blood samples were collected from healthy controls [n=9] following an 8-12 hours fast. Erythrocytes were purified twice by Boyum gradient. Erythrocytes were further sub-fractionated into young (y) (1.07-1.09 g/ml), mid (m) (1.09- 1.11 g/ml), and old (o) (1.11-1.12 g/ml) age cells by using discontinuous Percoll gradient (35%, 40%, 45%, 50%, 55%, 65%, 80%, and 100%) and total RNA extracted. MiRNA-144 and miRNA-451 were quantified in y, m, and o age E sub-fractions by qRT-PCR. Results: MiRNA-451 expression was 82210.8271, 130922.476, and 149554.364 in y, m, and o cells, respectively. MiRNA-144 expression in y cells was 18.6641092, m cells was 32.4413621, and o cells was 57.8118394 These results showed that o cells expressed both miRNA-144 and miRNA-451 more than that of m, and y cells. Conclusion: The findings of this study showed that miRNAs expression differ in sub-fractionated erythrocytes. This study suggests that miRNA-144 and miRNA-451 have the potential to be used as biomarkers of RBC aging.

Angiotensinogen import in isolated proximal tubules: evidence for mitochondrial trafficking and uptake

The renal proximal tubules are a key functional component of the kidney and express the angiotensin precursor angiotensinogen; however, it is unclear the extent that tubular angiotensinogen reflects local synthesis or internalization. Therefore, the current study established the extent to which angiotensinogen is internalized by proximal tubules and the intracellular distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with radiolabeled 125I-angiotensinogen for 2 h at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenous 125I-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus (60 ± 7%), endoplasmic reticulum (4 ± 0.5%), and cytosol (15 ± 4%; n = 4). Subsequent studies determined whether mitochondria directly internalized 125I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalized 125I-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min−1·mg protein−1; n = 3). Lastly, unlabeled angiotensinogen (100 nM) competed for 125I-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria (60 ± 2 vs. 16 ± 13%; P < 0.05, n = 3). Collectively, our data demonstrate angiotensinogen import and subsequent trafficking to the mitochondria in proximal tubules. We conclude that this pathway may constitute a source of the angiotensinogen precursor for the mitochondrial expression of angiotensin peptides.

Evidence for a mitochondrial angiotensin-(1–7) system in the kidney

Evidence for an intracellular renin-angiotensin system (RAS) in various cell organelles now includes the endoplasmic reticulum, nucleus, and mitochondria (Mito). Indeed, angiotensin (ANG) AT1 and AT2 receptor subtypes were functionally linked to Mito respiration and nitric oxide production, respectively, in previous studies. We undertook a biochemical analysis of the Mito RAS from male and female sheep kidney cortex. Mito were isolated by differential centrifugation followed by a discontinuous Percoll gradient and were coenriched in Mito membrane markers VDAC and ATP synthase, but not β-actin or cathepsin B. Two distinct renin antibodies identified a 37-kDa protein band in Mito; angiotensinogen (Aogen) conversion was abolished by the inhibitor aliskiren. Mito Aogen was detected by an Aogen antibody to an internal sequence of the protein, but not with an antibody directed against the ANG I N terminus. ANG peptides were quantified by three direct RIAs; mitochondrial ANG II and ANG-(1–7) contents were higher compared with ANG I (23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg protein; P < 0.01, n = 3). 125I-ANG I metabolism primarily revealed the formation of 125I-ANG-(1–7) in Mito that reflects the endopeptidases neprilysin and thimet oligopeptidase. Last, immunoblot studies utilizing the ANG-(1–7)/Mas receptor antibody revealed the protein in isolated Mito from sheep renal cortex. Collectively, the current data demonstrate that Mito actively metabolize the RAS precursor protein Aogen, suggesting that ANG-(1–7) may be generated within Mito to establish an intramitochondrial RAS tone and contribute to renal mitochondrial function.