Nitric oxide production by glomerular podocytes

Abstract

Nitric Oxide (NO), a potent vasodilator and vital signaling molecule, has been shown to contribute to the regulation of glomerular ultrafiltration. However, whether changes in NO occur in podocytes during the pathogenesis of salt-sensitive hypertension has not yet been thoroughly examined. We showed here that podocytes produce NO, and further hypothesized that hypertensive animals would exhibit reduced NO production in these cells in response to various paracrine factors, which might contribute to the damage of glomeruli filtration barrier and development of proteinuria. To test this, we isolated glomeruli from the kidneys of Dahl salt-sensitive (SS) rats fed a low salt (LS; 0.4% NaCl) or high salt (HS; 4% NaCl, 3 weeks) diets and loaded podocytes with either a combination of NO and Ca²⁺ fluorophores (DAF-FM and Fura Red, respectively) or DAF-FM alone. Changes in fluorescence were observed with confocal microscopy in response to adenosine triphosphate (ATP), angiotensin II (Ang II), and hydrogen peroxide (H₂O₂). Application of Ang II resulted in activation of both NO and intracellular calcium ([Ca²⁺]_i) transients. In contrast, ATP promoted [Ca²⁺]_i transients, but did not have any effects on NO production. SS rats fed a HS diet for 3 weeks demonstrated impaired NO production: the response to Ang II or H₂O₂ in podocytes of glomeruli isolated from SS rats fed a HS diet was significantly reduced compared to rats fed a LS diet. Therefore, glomerular podocytes from hypertensive rats showed a diminished NO release in response to Ang II or oxidative stress, suggesting that podocytic NO signaling is dysfunctional in this condition and likely contributes to the development of kidney injury.

Keywords: Angiotensin II; DAF-FM; Dahl salt-sensitive rat; Hydrogen peroxide; Hypertension; Nitric oxide.

Fig. 1

Method of glomeruli isolation and confocal imaging. (A) The kidneys are excised and glomeruli are isolated from renal cortex by differential sieving as reported previously [38; 49]. Shown is an image of freshly isolated decapsulated glomeruli, which are subsequently loaded with fluorescent calcium (Fura Red) and NO (DAF-FM) dyes to perform confocal imaging. Scale bar is 80 μm. (B) Representative images of a rat glomerulus stained with DAF-FM (green pseudocolor) and Fura Red (red pseudocolor). Image of the same glomerulus taken with transmitted light is also shown. ROI – single Region of Interest (one podocyte) used for analysis.

Fig. 2

Ang II evokes NO production and an increase in [Ca²⁺]_i in glomerular podocytes of SS rats. (A) Representative transients of [Ca²⁺]_i dynamics in the podocytes of the Fura Red loaded SS rat glomeruli in response to application of ATP and Ang II. (B) DAF-FM transient increase demonstrating NO production in response to the same agents. ATP application produces a Ca²⁺ transient without affecting NO production in glomeruli podocytes. In contrast, application of Ang II promotes both Ca²⁺ and NO level elevation (note that a decrease in Fura Red signal shows elevation of intracellular Ca²⁺).

Fig. 3

Ang II and H₂O₂ evoke NO production in glomeruli podocytes. (A) Confocal imaging of glomeruli before (upper panel) and after (lower panel) Ang II application (merged with transmitted light). Scale bars are shown. DAF-FM transient increase demonstrating elevated levels of NO in response to 20 μM Ang II (B) or 5 μM H₂O₂ (C). Both Ang II- and H₂O₂-mediated NO production significantly decreased after pre-treatment of glomeruli with L-NAME (10 mM).

Fig. 4

Impaired NO signaling in glomeruli podocytes of SS rats upon the development of salt-sensitive hypertension. (A) Changes in podocytes DAF-FM fluorescence in response to Ang II (10 μM) application on glomeruli of SS rats fed a LS (0.4% NaCl; black) and HS (4% NaCl, 3 weeks; red) diets. (B) Total NO production in response to Ang II stimulation calculated as an integral of DAF-FM transient for the 300 sec time interval (N≥5; n≥38 for each group; *P<0.05). (C) Changes in podocytes DAF-FM fluorescence in response to H₂O₂ (10 μM) in SS rats fed LS or HS diets. (D) Graph summarizing changes in H₂O₂-mediated NO production in glomeruli podocytes (N≥4; n≥30 for each group; *P<0.05).

Fig. 5

Changes in glomerular volume and permeability during the activation of NO signaling cascades in freshly isolated rat glomeruli. (A) Glomerular volume dynamics in response to changes in osmotic pressure (BSA changes from 5 to 1%) in control (left) or DETA NONOate pretreated (right) glomeruli. The line/symbol represents glomerular volume profile (each symbol is the size of a certain z-scan) before (black; t=0) and after (red; t=4min) change in osmotic pressure. (B) An example of glomerular volume change observed by confocal microscopy (shown are images in 5% BSA and after solution change to 1% BSA). (C) A summary of glomerular volumes (which reversibly correlates with the permeability of glomeruli filtration barrier) in control (vehicle) and DETA NONOate pretreated groups (N≥4; n≥12 for each group; *P<0.05).