Western blot analysis of heart and kidney homogenates from wild type (WT) and MasR-KO mice.

<p>The predicted molecular weight of MasR is about 37 kDa. As observed, the assayed antibodies generated different immunoreactivity patterns. In both cases there were no differences in the band patterns obtained with tissues from WT and MAS-KO mice. The membranes are representative of n = 4.</p

Immunohistochemistry in heart and kidney from wild-type (WT) and MasR-KO mice.

<p>Negative controls performed by omitting the primary antibody demonstrated minimal background immunostaining in heart (A) and kidney (B) sections. (A) In heart sections, NLS-1531 antibody stained predominantly the cytoplasm of cardiomyocytes with similar intensity in WT and MasR-KO hearts. The AAR-013 antibody stained the cardiomyocytes nucleus and weaker staining was observed in their cytoplasm. The same pattern was found in heart sections of MasR-KO mice. (B) In kidney sections, for NLS-1531 antibody, staining was mostly restricted to cytoplasm of numerous tubules cells with similar intensity in WT and MasR-KO kidneys. Antibody AAR-013 stained most intensely tubules cells nucleus and weaker staining was observed in their cytoplasm. Glomeruli were also stained positively. The same pattern was found in kidney sections of MasR-KO. Preincubation of the AAR-013 antibody with the blocking peptide provided by the vendors eliminated the immunohistochemical nuclear staining both in WT and MasR-KO mice kidney sections. Images are representative of n = 3. Bar, 50 μm.</p

Western blotting of HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag and three MasR antibodies.

<p>Cells were transfected with varying amounts of a plasmid encoding the c-myc-tagged MasR (0.2 and 0.5 μg of pcDNA 3.1/c-myc-MasR).The anti-c-myc antibody detected a band of 37 kDa whose intensity increased as higher amount of DNA was transfected (arrow). Additionally, a ladder of higher molecular weight (MW) immunoreactive bands could be observed (asterisk). sc-54682 antibody reproduced the band pattern obtained with the c-myc antibody (arrow and asterisk). sc-135063 antibody revealed multiple bands close to 37 kDa with one of them of increasing intensity as higher amount of DNA was transfected (arrow) but did not produce a ladder of higher MW immunoreactive bands. High unspecific staining was attained with sc-135063 antibody. AAR-013 antibody revealed a pattern of immunoreactivity not comparable to those obtained with the other antibodies. Detection of β-tubulin was used as a protein loading control. The membranes are representative of 3 independent experiments.</p

Detection of MasR mRNA in heart and kidney from wild type and MasR-KO mice.

<p>Representative agarose gel (n = 3–5) showing reverse transcription polymerase chain reaction (RT-PCR) product obtained from hearts (A) and kidneys (B) of wild type (WT) and MasR-KO mice. MasR mRNA was undetectable in heart and kidney of MasR-KO mice. 18S ribosomal RNA (18S rRNA) was used as housekeeping gene.</p

Immunofluorescence studies in HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag antibody.

<p>Images of fluorescence signal corresponding to secondary cyanine (Cy3)-conjugated antibody (A), nuclear Hoechst 33258 staining (B), merged images (C) and bright field (D) are shown. The intense immunostaining obtained with the anti c-myc tag antibody indicated strong expression of MasR in HEK293T cells after transfection with the pcDNA 3.1/c-myc-MasR construct. A significant amount of the total MasR pool was present at the plasma membrane. No c-myc staining was observed in cells transfected with the empty vector pcDNA 3.1. Images are representative of 3 independent experiments.</p

Renal enzymatic activity of ACE, ACE2 and the ACE/ACE2 ratio.

<p>(A) ACE enzymatic activity. (B) ACE2 enzymatic activity. (C) ACE/ACE2 ratio. Data are means ± SEM. NS: normal salt diet. LS: low salt diet. HS: high salt diet. *P<0.05 vs. NS; + P < 0.05 vs. HS. (NS n = 12, LS n = 10, HS n = 9).</p

Histological analysis.

<p>(A) Photomicrographs of kidney sections stained with Periodic Acid Schiff (PAS) to determine glomerulosclerosis score (percentage of the PAS-positive in glomerular tuft area) and morphometric changes in the groups. (B) Photomicrographs of kidney sections stained with Picrosirius Red to determine interstitial collagen deposition. Data are represented as mean ± SEM. NS: normal salt diet. LS: low salt diet. HS: high salt diet. Scale bar: 20 μm.</p

Analysis of megalin and cubilin renal protein expression.

<p>(A) Megalin. (B) Cubilin. Data are means ± SEM. (NS) normal salt diet. LS: low salt diet. HS: high salt diet. *P<0.05 vs. NS; + P < 0.05 vs. HS. (n = 6 / group).</p

Evaluation of the urinary protein loss.

<p>24-h urine samples from SHRs were subjected to 12% SDS-PAGE and silver stained. NS: normal salt diet. LS: low salt diet. HS: high salt diet.</p

Glomeruli ultrastructural examination.

<p>Transmission electron microscopy images show the ultrastructural pedicels (fp) and slit diaphragms (SD, arrows) from the podocytes. Images A and B show the normal podocyte structure from Normal and Low salt group respectively, with SD integrity. Image C shows that the high salt group has lost the morphological integrity of its podocyte processes (fusion/effacement*) along with the slit diaphragms (black arrow head). BM, basement membrane; EC, endothelial cell.</p