Renal Effects and Underlying Molecular Mechanisms of Long-Term Salt Content Diets in Spontaneously Hypertensive Rats.

Several evidences have shown that salt excess is an important determinant of cardiovascular and renal derangement in hypertension. The present study aimed to investigate the renal effects of chronic high or low salt intake in the context of hypertension and to elucidate the molecular mechanisms underlying such effects. To this end, newly weaned male SHR were fed with diets only differing in NaCl content: normal salt (NS: 0.3%), low salt (LS: 0.03%), and high salt diet (HS: 3%) until 7 months of age. Analysis of renal function, morphology, and evaluation of the expression of the main molecular components involved in the renal handling of albumin, including podocyte slit-diaphragm proteins and proximal tubule endocytic receptors were performed. The relationship between diets and the balance of the renal angiotensin-converting enzyme (ACE) and ACE2 enzymes was also examined. HS produced glomerular hypertrophy and decreased ACE2 and nephrin expressions, loss of morphological integrity of the podocyte processes, and increased proteinuria, characterized by loss of albumin and high molecular weight proteins. Conversely, severe hypertension was attenuated and renal dysfunction was prevented by LS since proteinuria was much lower than in the NS SHRs. This was associated with a decrease in kidney ACE/ACE2 protein and activity ratio and increased cubilin renal expression. Taken together, these results suggest that LS attenuates hypertension progression in SHRs and preserves renal function. The mechanisms partially explaining these findings include modulation of the intrarenal ACE/ACE2 balance and the increased cubilin expression. Importantly, HS worsens hypertensive kidney injury and decreases the expression nephrin, a key component of the slit diaphragm

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

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

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

Analysis of nephrin and podocin renal protein expression.

<p>(A) Nephrin. (B) Podocin. 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 LS. (N = 6 / group).</p

Parameters of renal function.

<p>Values are represented as the mean± SEM. NS: normal salt diet. LS: low salt diet. HS: high salt diet. GFR: glomerular filtration rate</p><p>*P<0.05 vs. NS</p><p>+ P < 0.05 vs. HS</p><p># P < 0.05 vs LS.</p><p>Parameters of renal function.</p

Analysis of ACE, ACE2 expression and ACE/ACE2 protein ratio.

<p>(A) ACE kidney expression. (B) ACE2 kidney expression. C: ACE/ACE2 protein 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; # P < 0.05 vs LS.</p

Morphologic characteristics of the kidneys.

<p>Values are mean ± SEM. SBP: systolic blood pressure. NS: normal salt diet. LS: low salt diet. HS: high salt diet. SBP at weaning: subsample of 8 rats.</p><p>* P<0.05 vs. NS</p><p>+ P < 0.05 vs. HS</p><p># P < 0.05 vs LS.</p><p>(n = 6 / group for histological parameters).</p><p>Morphologic characteristics of the kidneys.</p