Abstract 106: Signaling Mechanisms Affecting Nitric Oxide Production in Glomerular Podocytes

Abstract

While Nitric Oxide (NO), a potent vasodilator and vital signaling molecule, has been shown to contribute to the regulation of glomerular ultrafiltration, its role in podocytes during the pathogenesis of salt-sensitive hypertension has not yet been thoroughly examined. Recent studies have demonstrated that the deficiency of eNOS (the enzyme responsible for synthesizing NO) exacerbates renal injury and accelerates development of proteinuria and glomerulosclerosis. Considering this, we hypothesized that the podocytes of hypertensive animals would exhibit reduced NO production in response to various paracrine factors and this directly contributes to proteinuria. To test this, we isolated glomeruli from the kidneys of Dahl salt-sensitive (SS) rats fed either a high salt (HS; 4% NaCl, 3 weeks) or low salt (LS; 0.4% NaCl) diet and loaded podocytes with a combination of NO and Ca 2+ ionophores (DAF-FM and Fura Red, respectively). Changes in fluorescence were observed with the use of confocal microscopy in response to adenosine triphosphate (ATP), angiotensin II (Ang II), and H 2 O 2 . Application of Ang II or H 2 O 2 resulted in activation of both NO and [Ca 2+ ] i ; fluorescent transients which were significantly elevated in the soma and foot processes of podocytes of LS fed rats. In contrast, ATP specifically activated only changes in [Ca 2+ ] i , but did not have any effects on NO production. Ang II treatment also caused hypertrophy of the podocytes, whereas ATP had no effect on cell volume (41.1±7.7 vs. 0.1±3.6% increase for Ang II and ATP, respectively; P <0.05). Collectively, our results show that in contrast to [Ca 2+ ] i , which is modulated by all studied paracrine molecules, NO is produced by podocytes only in response to Ang II and H 2 O 2 , but not ATP. SS rats fed a HS diet for 3 weeks demonstrated impaired NO production; the response to Ang II or H 2 O 2 on HS contained only 23.7±6.6 and 43.4±28.4% of total effects on LS, respectively ( P <0.05). Therefore, when fed a HS diet, SS podocytes had an impaired NO response to Ang II or oxidative stress, suggesting that NO signaling is dysfunctional and likely contributes to the development of kidney injury.