Alterations in renal Na,K-ATPase activity and protein expression in rat models of pressure and volume overload

Figures

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  Fig. 1. Histological demonstration of collagen deposition (pink color) by Van Gieson staining (Panel A). Black arrows point to vascular deposition of collagen, while white arrows point to the deposition of collagen in the area of the glomerular basement membrane. Bar: 200 μm. Representative western blot images and zymogram (Panel B). Quantification of collagen deposition (Panel C). Renal thiobarbituric acid reactive substances (TBARS) levels (Panel D). Protein levels of renal protein kinase C delta (PKC−δ, Panel E). Protein levels of renal neutrophil gelatinase−associated lipocalin (NGAL, Panel F). Matrix−metalloproteinase−2 activity (MMP−2, Panel G). Results are presented for Hannover Sprague Dawley rats (HAN), transgenic rats TGR(mREN2)27 (TGR), with or without aortocaval fistula (ACF) operation (n=6–8/each group). ACF was induced in 8−week−old animals, and they were sacrificed at 11 weeks of age. Glyceraldehyde 3−phosphate dehydrogenase (GAPDH) was used as loading control for PKC−δ and NGAL protein normalization. Data represent means ± standard deviations. For statistical analyses, two−way ANOVA was applied to determine the effect of two factors − genetic modification (GEN.) and ACF,…

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  Fig. 2. Kinetic parameters of the Na, K−ATPase enzyme during activation by the ATP substrate in Hannover Sprague Dawley rats (HAN) and TGR(mREN2)27 rats (TGR), both sham−operated (C) and subjected to aortocaval fistula (ACF) surgery. ACF was induced in 8−week−old animals, and they were sacrificed at 11 weeks of age. The parameter Vmax represents the maximal velocity of enzyme reaction, the Km value refers to the concentration of ATP necessary for half maximal activation of the enzyme. Data represent means ± standard deviations, (n = 6–8 animals/group, technical replicates 3/animal). For statistical analyses, two−way ANOVA was applied to determine the effect of two factors − genetic modification (GEN.) and ACF, as well as their interaction (INT.). Statistical significance: ***p < 0.001, *****p < 0.0001. To identify significant differences between individual groups, Tukey’s post−hoc test was used. Statistical significance: a: p < 0.05 vs. HAN−C, b: p < 0.05 vs. TGR−C.

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  Fig. 3. Activation of the Na, K−ATPase in sham−operated rats by low concentrations of energy substrate ATP. Inset: activation of the enzyme in the whole investigated concentration range in sham−operated Hannover Sprague Dawley (HAN, n = 8) and transgenic rats TGR(mREN2)27 (TGR, n = 8). The animals underwent a sham operation at 8 weeks of age and were sacrificed at 11 weeks of age. Data represent means ± standard deviations.

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  Fig. 4. Activation of the Na, K−ATPase in aortocaval fistula (ACF)−operated rats by low concentrations of energy substrate ATP. Inset: activation of the enzyme in the whole investigated concentration range in ACF−operated Hannover Sprague Dawley (HAN, n = 6) and transgenic rats TGR(mREN2) 27 (TGR, n = 6). ACF was induced in 8−week−old animals, and they were sacrificed at 11 weeks of age. Data represent mean ± standard deviations.

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  Fig. 5. Activation of the Na,K−ATPase in sham− and ACF−operated rats by low concentrations of energy substrate ATP. Inset: activation of the enzyme in the whole investigated concentration range in Hannover Sprague Dawley (HAN, panel A) and transgenic rats TGR(mREN2)27 (TGR, panel B). ACF was induced in 8−week−old animals, and they were sacrificed at 11 weeks of age. Data represent mean ± standard deviations, (n = 6–8 each group).

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  Fig. 6. Representative western blot images (A) and protein levels of renal Na, K−ATPase subunits α1 (Panel B) and β1 (Panel C, D), measured in Hannover Sprague Dawley rats (HAN), transgenic rats TGR(mREN2)27 (TGR), with or without aortocaval fistula (ACF) operation (n = 6–8 in each group). ACF was induced in 8−week−old animals, and they were sacrificed at 11 weeks of age. Beta−actin was used as loading controls for protein normalization. Data represent means ± standard deviations. For statistical analyses, two−way ANOVA was applied to determine the effect of two factors − genetic modification (GEN.) and ACF, as well as their interaction (INT.). Statistical significance: ***p<0.001, *****p<0.0001. To identify significant differences between individual groups, Tukey’s post−hoc test was used. Statistical significance: a: p<0.05 vs. HAN−C, b: p<0.05 vs. TGR−C.