Inducible Nitric-oxide Synthase Is an Important Contributor to Prolonged Protective Effects of Ischemic Preconditioning in the Mouse Kidney

Abstract

Ischemic preconditioning renders the mouse kidney resistant to subsequent ischemia. Understanding the mechanisms responsible for ischemic preconditioning is important for formulating therapeutic strategies aimed at mimicking protective mechanisms. We report that the resistance afforded by 30 min of bilateral kidney ischemia persists for 12 weeks after preconditioning. The protection is reflected by improved postischemic renal function, reduced leukocyte infiltration, reduced postischemic disruption of the actin cytoskeleton, and reduced postischemic expression of kidney injury molecule-1 (Kim-1). The protection is observed in both BALB/c and C57BL/6J strains of mice. Thirty minutes of prior ischemia increases the expression of inducible nitric-oxide synthase (iNOS) and endothelial NOS (eNOS) and the expression of heat shock protein (HSP)-25 and is associated with increased interstitial expression of ␣-smooth muscle actin (␣-SMA), an indication of long term postischemic sequelae. Treatment with N-nitro-L-arginine (L-NNA), an inhibitor of NO synthesis, increases kidney susceptibility to ischemia. Gene deletion of iNOS increases kidney susceptibility to ischemia, whereas gene deletion of eNOS has no effect. Pharmacological inhibition of NOS by L-NNA or L-N6-(1-iminoethyl) lysine (L-NIL, a specific inhibitor of iNOS) mitigates the kidney protection afforded by 30 min of ischemic preconditioning. Fifteen minutes of prior ischemic preconditioning, which does not result in the disruption of the actin cytoskeleton, impairment of renal function, increased interstitial ␣-SMA, or increased iNOS or eNOS expression, but does increase HSP-25 expression, partially protects the kidney from ischemia on day 8 via a mechanism that is not abolished by L-NIL treatment. Thus, iNOS is responsible for a significant component of the long term protection afforded the kidney by ischemic preconditioning, which results in persistent renal interstitial disease, but does not explain the preconditioning seen with shorter periods of ischemia.

Guanylyl cyclase is an ATP sensor coupling nitric oxide signaling to cell metabolism

“…Indeed, NO and cGMP regulate mitochondrial biogenesis in response to hypothermic stress and interruption of that signaling mechanism disrupts body energy balance (17). Moreover, NO activation of sGC and the associated accumulation of [cGMP] i contribute to metabolic remodeling important in ischemic preconditioning in the cardiovascular and central nervous systems (4,5). Together, the coordinated reduction in cell functions demanding energy and increase in mechanisms supplying energy mediated by NO and cGMP improve steady-state ATP levels (35) and survival (36)(37)(38) in the context of a metabolic challenge.…”

“…Beyond vascular smooth muscle relaxation associated with improved tissue perfusion (4)(5)(6), NO also regulates the supply of cellular energy in the face of a metabolic challenge. Thus, NO and the associated production of cGMP decrease the transport, availability through glycogenoloysis, and metabolic activation of glucose (9,10).…”

“…Metabolic stress, such as ischemia, stimulates NO synthases to produce NO from arginine (3). In turn, NO regulates the supply of energy by improving the delivery of substrates and oxygen to deprived tissues (4)(5)(6), regulating the intracellular delivery of metabolic substrates (7)(8)(9)(10), selection of substrates that support metabolism (6,(10)(11)(12)(13)(14), oxidation of metabolic substrates (15), generation of ATP by improving metabolic efficiency (16), and biogenesis of mitochondria (17). Conversely, NO reduces the demand for ATP by decreasing energyconsuming cell functions, for example, by reducing the activity of the contractile apparatus in muscle cells (ref.…”

Orchiectomy Attenuates Post-ischemic Oxidative Stress and Ischemia/Reperfusion Injury in Mice

“…Thirty minutes of bilateral ischemia in non-orchiectomized mice results in the disruption of most of tubular cells in the outer medulla leading to kidney dysfunction (8,24,25,42), whereas the same period of ischemia in orchiectomized mice does not induce extensive cellular death. In the present study, 30 min of ischemia in orchiectomized mice produced small amounts of free radicals as seen in Fig.…”

Atherosclerotic renal artery stenosis is associated with elevated cell cycle arrest markers related to reduced renal blood flow and postcontrast hypoxia

“…A growing body of evidence indicates that repeated ischemia and activation of chronic injury signaling pathways may induce natural defenses such as bioenergetic downregulation and temporary cell cycle arrest [11,41]. These defenses may be the substrate for 'ischemic preconditioning' that can protect the kidney during subsequent injury or ischemic stress [42,43]. While serum creatinine is frequently used to define AKI, it is recognized as an insensitive and delayed marker to identify renal injury [44].…”