Levels of cAMP.

<p>Levels of cAMP were decreased in Ang II treated AEC II cells by 0.17 folds compared to the control group. * P < 0.05 As compared to control group. CT—Control. Ang II—Angiotensin II. The bars represent mean ± SEM.</p

Effect of Ang II on AFC.

<p>(A) % Alveolar fluid clearance of the initial instilled volume was decreased in the Ang II groups in a dose dependent manner, from 8.6% ± 0.19 in control rats to 6.66% ± 0.13, 6.15% ± 0.11, 5.03% ± 0.31, 4.42% ± 0.29 and 5.25% ± 0.23 in Ang II (10⁻¹⁰ M, 10^-9M, 10⁻⁸ M, 10⁻⁷ M and 10⁻⁶ M) respectively. * P<0.001 As compared to control group; ** P<0.05 As compared to the rest of 10⁻¹⁰ M and 10⁻⁹ M Ang II treated groups. CT—Control. The bars represent mean ± SEM. (B) The albumin movement across the alveolar-capillary barrier did not differ significantly among the study groups indicating that the barrier was intact. CT—Control. The bars represent mean ± SEM.</p

A comparable scheme of AFC under normal vs. Ang II stimulated conditions.

<p>Under normal conditions Na+ is extruded out of the alveolar airspace by apical epithelial Na+ channels (ENaC), specifically highly selective cation channels composed of α, β and γ subunits (HSC) and basolateral Na,K-ATPase pump with water following osmoticaly, Whereas Ang II stimulation down regulated cAMP levels in AEC II, via AT1 receptors triggering, thus leading to the decrease of the two important AFC players; αNa,K-ATPase and the HSC, and an increase of the NSC (non-selective cation channels composed of α subunit alone), with a resultant impairment of sodium reabsorption and conceivable AFC decrease.</p

AT₁ levels.

<p>(A) A semi-quantative measure of AT1 protein levels in AEC II by western blotting; AT1 levels were significantly increased by 1.67 fold after Ang II administration. *P<0.0001 as compared to control group. CT—Control. Ang II—Angiotensin II. AT1—Angiotensin-II receptor type 1. The bars represent mean ± SEM. (B) An immunohestochemical staining of AT1 receptor in AEC II cells treated or untreated with Ang II. The representative figure showing stronger staining of AT1 in the Ang II treated group compared to the control. CT—Control. Ang II—Angiotensin II.</p

αNa,K-ATPase levels.

<p>(A) Na,K-ATPase α1 subunit levels in whole cell AEC II were measured by western blotting. Ang II administration decreased Na,K-ATPase levels by 0.65 folds compared to the control group. * P < 0.001 As compared to control group. CT—Control. Ang II—Angiotensin II. The bars represent mean ± SEM. (B) A representative Immunofluorescence staining of α1-Na,K-ATPase shows a shift of the protein membrane localization to internal organelles following Ang II administration. CT—Control. Ang II—Angiotensin II.</p

Different interventions effect on AFC.

<p>(A) Losartan restored Ang II effect on AFC from 5.25%±0.23 to 8.1%±0.13. AFC was not different in both losartan treated groups. * P<0.001 As compared to control group treated with Ang II. PD123319, AT₂ receptor antagonist, decreased AFC in both AngII treated (n = 4) and untreated groups (n = 4) (6.54%±0.2 and 6.51%±0.2 respectively). $ P<0.05 as compared to Ang II group, & P<0.001 as compared to control group. Ouabain, the Na,K-ATPase blocker, significantly inhibited AFC in both control and Ang II treated rat lungs (5.9% ± 0.4 and 4.4% ± 0.2 respectively). + P<0.05 as compared to control rat lungs treated with ouabain alone. Amiloride, the sodium channel blocker, significantly reduced AFC in both control and Ang II treated rats as compared to untreated lungs (a 5.6% ± 0.2 and 5.01 ± 0.2 respectively). However, AFC was similar in the two Amiloride treated groups. Activating the adrenergic pathway by norepinephrine 10^-6M increased the clearance percentage to 14.12% ± 1.8, when compared to control 8.6% ± 0.19. But when Ang II was also added, NE effect was abolished (7.3% ± 0.6). # P<0.05 as compared to control rat lungs treated with norepinephrine alone. ## P<0.0001 as compared to AngII group. CT—Control. Ang II—Angiotensin II. NE—Norepinephrine. The bars represent mean ± SEM. (B) The albumin movement across the alveolar-capillary barrier did not differ significantly among the study groups indicating that the barrier was intact. CT—Control. Ang II—Angiotensin II. Los—Losartan. PD—PD123319. Ouab—Ouabain. Amil—Amiloride. NE—Norepinephrine. The bars represent mean ± SEM.</p

Nephroprotective Effect of Heparanase in Experimental Nephrotic Syndrome

<div><p>Background</p><p>Heparanase, an endoglycosidase that cleaves heparan sulfate (HS), is involved in various biologic processes. Recently, an association between heparanase and glomerular injury was suggested. The present study examines the involvement of heparanase in the pathogenesis of Adriamycin-induced nephrotic syndrome (ADR-NS) in a mouse model.</p><p>Methods</p><p>BALB/c wild-type (<i>wt</i>) mice and heparanase overexpressing transgenic mice (<i>hpa</i>-TG) were tail-vein injected with either Adriamycin (ADR, 10 mg/kg) or vehicle. Albuminuria was investigated at days 0, 7, and 14 thereafter. Mice were sacrificed at day 15, and kidneys were harvested for various analyses: structure and ultrastructure alterations, podocyte proteins expression, and heparanase enzymatic activity.</p><p>Results</p><p>ADR-injected <i>wt</i> mice developed severe albuminuria, while ADR-<i>hpa</i>-TG mice showed only a mild elevation in urinary albumin excretion. In parallel, light microscopy of stained cross sections of kidneys from ADR-injected <i>wt</i> mice, but not <i>hpa</i>-TG mice, showed mild to severe glomerular and tubular damage. Western blot and immunofluorescence analyses revealed significant reduction in nephrin and podocin protein expression in ADR-<i>wt</i> mice, but not in ADR-<i>hpa</i>-TG mice. These results were substantiated by electron-microscopy findings showing massive foot process effacement in injected ADR-<i>wt</i> mice, in contrast to largely preserved integrity of podocyte architecture in ADR-<i>hpa</i>-TG mice.</p><p>Conclusions</p><p>Our results suggest that heparanase may play a nephroprotective role in ADR-NS, most likely independently of HS degradation. Moreover, <i>hpa</i>-TG mice comprise an invaluable <i>in vivo</i> platform to investigate the interplay between heparanase and glomerular injury.</p></div

Adriamycin causes renal damage in wild type mice but not in <i>hpa</i>-TG mice.

<p><i>wt</i> or <i>hpa</i>-TG mice were injected with Adriamycin (ADR) or served as control (sham). Two weeks post injection, the animals were sacrificed. Representative images of H&E <b>(A)</b> and Masson’s trichrome <b>(B)</b> staining of paraffin-embedded kidney sections from various experimental groups (scale bar = 100 μm). n = 6 mice per each experimental group, from three independent experiments.</p

<i>hpa</i>-TG mice are protected from Adriamycin-induced albuminuria and nephrotic syndrome.

<p><b>(A)</b> Male wild type BALB/c mice (<i>wt</i>) and <i>hpa</i>-TG mice were injected with Adriamycin (ADR) or kept as control (sham). Prior to injection (week 0) or one and two weeks post injection, urine was collected for 24h and used to determine albumin/creatinine ratio concentrations as described (n = 7 per each experimental group). **, P<0.001 vs. <i>hpa</i>-TG sham; ***, P<0.0001 vs. <i>wt</i> sham. <b>(B)</b> Blood samples were drawn from cardiac puncture at the day of sacrifice. Sera were analyzed for albumin and cholesterol (n = 13, 9, 12, and 11 for <i>wt</i> sham, <i>wt</i> ADR, <i>hpa</i>-TG sham, and <i>hpa</i>-TG ADR mice, respectively). **, P<0.001 vs. <i>wt</i> sham.</p

Heparanase protects podocytes from Adriamycin induced injury.

<p><i>wt</i> or <i>hpa</i>-TG mice were injected with Adriamycin (ADR) or served as control (sham). Two weeks post injection, the animals were sacrificed. <b>(A)</b> Representative images of transmission electron microscopy of ultrathin sections of kidney tissue. Magnification X15 000; scale bar = 1μm. <b>(B)</b> Quantification of foot process width (n = 12 glomeruli per each wt group, 13 for <i>hpa</i>-TG, and 11 for <i>hpa</i>-TG ADR group, obtained from 6, 3, 4, and 5 animals, respectively). *, P<0.01 vs. all other groups.</p