Renal Glomerular Mitochondria Function in Salt-Sensitive Hypertension

Figures

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  FIGURE 1. Study protocol and development of the salt-sensitive (SS) hypertension in the Dahl SS rats. (A) Schematic representation of the experimental protocol used to induce SS hypertension in Dahl SS rats. (B) Graphs illustrating the observed increase in systolic blood pressure (systolic BP, left) and two kidney to body weight ratio (middle panel, 2 k/BW) in the NS diet fed group compared to a HS diet fed group (right). (C) Representative histological images showing cortical renal tissues (20×) and medullary tissues (10×) from NS and HS diet fed animals stained with Masson’s trichrome. Graph illustrates summarized glomerular damage score (glomeruli examples are provided below for each experimental group). All data were compared using a one-way ANOVA with Holm–Sidak post hoc test. In Graphs (B), each data point represents a single measurement from an experimental animal at the end of the protocol after the NS or HS challenges. For the glomerular damage score (C), each point is an average of 100 glomeruli blindly scored in the renal tissue of each animal. NS, normal salt; HS, high salt.

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  FIGURE 2. Renal mitochondria biogenesis and electron transport chain (ETC) complexes expression and activity during salt-sensitive (SS) hypertension. (A) Representative Western blot showing expression of PGC1α in the renal cortex of the HS and NS diet fed animals. Graph on the right summarizes densitometry values from the Western blot shown on the left combined with another (independent) Western run on other samples (data not shown). All data were normalized to GAPDH expression level. N = 7 individual rats tested in each group. (B) Mitochondrial DNA content in the renal cortex of rats fed a HS or a NS diet. Shown are results of qPCR for mitochondria genome encoded genes Nd1 and Nd6 (values normalized to nuclei-encoded Actb). (C) ETC Complex V activity as measured in the glomeruli isolated from NS and HS diet fed rats. Data were normalized to protein level, n = 5 animals per group. (D,E) Western blot analysis illustrating the expression of ETC complexes V, IV, III, II, and I in the renal cortex (D) and isolated glomeruli (E) from HS and NS diet fed Dahl SS rats. Graph on the right summarizes densitometry values (normalized to total protein as measured with Ponceau stain). NS, normal salt;…

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  FIGURE 3. Relative expression of the Atp5f1a, Cycs, Ppargc1a, and Cox4i1 genes assessed in the fraction of the glomeruli isolated from normal salt (NS) and high salt (HS) diet fed Dahl SS rats. Each point on the graph is a sample from a separate animal. P-values were obtained with t-tests, assuming unequal variances.

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  FIGURE 4. Mitochondrial respiration in the freshly isolated glomeruli during SS hypertension. (A) Oxygen consumption rate (OCR, pmol/min/mg protein) values obtained in a Seahorse experiment from glomeruli freshly isolated from the renal cortex of rats fed a high salt (HS) or normal salt (NS) diet. Shown are curves summarized from at least five rats per group, three to five replicates per rat; error bars are SEM. Right inset panel illustrates the experimental design. (B) Summary for basal respiration, ATP production, proton leak, maximal respiration, and reserve respiratory capacity as measured in the seahorse experiment shown in Graph. OCR is expressed in pmol/min/mg protein (A). Each data point is an average of at least three replicates per individual rat. An inset shows a typical preparation of isolated glomeruli. All data were compared using a one-way ANOVA with Holm–Sidak post hoc test. NS, normal salt; HS, high salt.

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  FIGURE 5. Structural and functional properties of glomerular mitochondria in salt-sensitive (SS) hypertension. (A) A schematic illustration of the imaging configuration, as well as representative images of the podocytes in the isolated glomeruli stained with mitoPY1 to visualize H2O2 in mitochondria (green), and Hoechst (blue) to label nuclei. Images were obtained with a 63× oil objective, NA 1.4. Dotted lines show podocyte body outlines (obtained from TL images). Graph on the right summarizes the fluorescence transients obtained from mitoPY1-stained podocytes of freshly isolated glomeruli in response to acute addition of 10 μM H2O2. Background auto fluorescence of mitoPY1 is shown (transient obtained in the absence of H2O2). N, five rats per group, at least four glomeruli per animal, with five to nine ROIs (podocytes) analyzed per glomerulus. Statistical analysis performed using one-way repeated measures ANOVA with Holm–Bonferroni for post hoc means comparison. (B) Representative electron microscopy images of the mitochondria ultrastructure in the podocyte of the glomeruli isolated from NS and HS diet fed rats. Scale bar is shown on the graph; asterisk denotes autophagosome formation;…

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  FIGURE 6. Suggested schematic illustration of the contribution of renal mitochondrial dysfunction to glomerular damage occurring in salt-sensitive (SS) hypertension.