5. Reactive oxygen species, shear stress and cilia function

ROS and NO have been implicated in sensing FSS in both vascular homeostasis and diseases [46]. ROS include collective oxygen (O2) radicals such as superoxide, O2 ˙ and hydroxyl radical, OH, and non-radical derivatives of O2, including hydrogen peroxide (H2O2) and ozone (O3). Several sources of ROS have been extensively described in the literature, in which the nicotidamine adenine dinucleotide phosphate (NADP) oxidase (Nox) has been described as one of the main cellular sources of ROS generation in endothelial cells under FSS [47].

Flow patterns and the magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady laminar or pulsatile) [48]. In addition to flow pattern, endothelial cells exposed to a prolonged laminar shear stress for more than 24 h display a reduced O2 ˙ formation and ROS levels [49]. ROS production is closely linked to NO generation: elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow patterns [48]. The low NO bioavailability is partially provoked by ROS reaction with NO to form peroxynitrite (ONOO), a key molecule that is implicated in oxidative and nitrosative damage [50]. NO can also take part in redox signaling by modifying proteins and lipids via cysteine Snitrosation and fatty acid nitration, respectively [51], in this respect affecting the regulation of the vascular system [52].

#### 5.1. Free radical signaling and primary cilia

Information related to primary cilium and free radical signaling emerges mainly from kidney research area. However, how ROS can regulate this mechanosensory organelle is not well described in the literature [17, 30, 53]. It is known that renal primary cilia protrude from the epithelial cell surface into the lumen detecting fluid flow and responding to diverse stimuli [12]. Indeed, several reports show that an alteration of primary cilia length is associated with acute and chronic kidney disease [16].

Information about primary cilia acting as an upstream regulator of ROS comes primarily from in vitro experiments, in which immortalized macula densa cell line (MMDD1) exposed to an increment in shear stress shows augmented NO production, this effect was blunted by silencing polaris protein in primary cilia using si-RNA methodology [54]. In addition, in isolated perfused juxtaglomerular apparatus preparations incubated with the diuretic furosemide (an inhibitor of Na-K-Cl cotransporter), an increase in tubular flow-induced NO production was observed. This suggests that the NO stimulatory effect is independent of Na+ concentration in the tubular fluid, as well as volume changes, suggesting a direct FSS-dependent regulation [30]. Also, the results elucidate that FSS can stimulate NO production independently of NaCl delivery to the macula densa. Therefore, these results indicate that the primary cilium acts as a mechanosensory organelle for FSS inside the nephron tubule via NO.

The process of disassembly observed here involves the termination of IFT and the inability of the oldest centriole to maintain or initiate the assembly of primary cilia under laminar shear stress [28]. The disassembly of cilia parallels a major rearrangement of the cytoskeleton and an

In the renal system, tubular flow and ROS act as potent modulators of epithelial kidney cell phenotype also by affecting the organization of the cytoskeleton and the brush border, changing cell polarity and modifying various cellular functions such as solute reabsorption and extracellular matrix remodeling [17]. Under oxidative stress, ROS directly induce the breakdown of the cell cytoskeleton, activate various cell death-associated signals and regulate elongation, shortening and release of cilia [45]. The mechanism and implications of this regu-

ROS and NO have been implicated in sensing FSS in both vascular homeostasis and diseases

radical, OH, and non-radical derivatives of O2, including hydrogen peroxide (H2O2) and ozone (O3). Several sources of ROS have been extensively described in the literature, in which the nicotidamine adenine dinucleotide phosphate (NADP) oxidase (Nox) has been described as

Flow patterns and the magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady laminar or pulsatile) [48]. In addition to flow pattern, endothelial cells exposed to a prolonged laminar shear stress for

closely linked to NO generation: elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow patterns [48]. The low NO bioavailability is partially provoked by ROS reaction with NO to form peroxynitrite (ONOO), a key molecule that is implicated in oxidative and nitrosative damage [50]. NO can also take part in redox signaling by modifying proteins and lipids via cysteine Snitrosation and fatty acid nitration, respectively [51], in this respect affecting the regulation

Information related to primary cilium and free radical signaling emerges mainly from kidney research area. However, how ROS can regulate this mechanosensory organelle is not well described in the literature [17, 30, 53]. It is known that renal primary cilia protrude from the epithelial cell surface into the lumen detecting fluid flow and responding to diverse stimuli [12]. Indeed, several reports show that an alteration of primary cilia length is associated with

˙ formation and ROS levels [49]. ROS production is

one of the main cellular sources of ROS generation in endothelial cells under FSS [47].

˙ and hydroxyl

5. Reactive oxygen species, shear stress and cilia function

[46]. ROS include collective oxygen (O2) radicals such as superoxide, O2

increase of acetylation of MT [18, 44].

96 Endothelial Dysfunction - Old Concepts and New Challenges

more than 24 h display a reduced O2

5.1. Free radical signaling and primary cilia

acute and chronic kidney disease [16].

of the vascular system [52].

lation are still unclear.

The opposite mechanism in which free radical species can regulate primary cilia function is showed mainly in renal ischemia/reperfusion (I/R) experiments. I/R setting is characterized by an increase in free radical species production [55]. Acute tubular necrosis induced by I/R on mice model resulted in changes in primary cilium length. Thus, primary cilium was shortened after 4 h and 1 day of ischemia versus non-ischemic control cells, an effect that was blunted after 16 days [16]. The oxidative stress from I/R derived injury is able to break down cell cytoskeleton and activate various cell death-associated signals, like cell autophagy [45]. As presented by Kim et al. [53], the treatment with the antioxidant molecule Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP) during the reperfusion (i.e., recovery) period of damaged kidneys accelerated the normalization of cilia length in experiments of I/R. Concomitantly, they also showed that MnTMPyP decrease oxidative stress and recover nephron tubule morphology, indicating that the ROS signals are an integral part of cilium length regulation. In addition, cultured kidney cells treated with H2O2 released a ciliary fragment into the extracellular medium. MnTMPyP treatment inhibited this deciliation process [17, 53]. Moreover, the extracellular signal-regulated kinase (ERK) inhibitor U0126 blocked the cilium elongation of normal and H2O2-treated cells [53]. Taken together, these observations show that primary cilia length can be regulated, at least in part, by H2O2 through an ERK-dependent pathway. Similar results were found related in acute kidney injury after hepatic I/R from liver transplantation or resection experiments in the kidney [56]. In particular, transient hepatic ischemia caused functional and histological kidney damage, including brush border loss of tubular epithelial cells and tubule atrophy. This cellular damage also induces a shortening and deciliation of kidney primary cilia via ROS/oxidative stress, suggesting that the presence of ciliary proteins in the urine could be a potential indication of kidney injury [17]. Therefore, remote organ injury model can increase the content of O2 , and H2O2 subsequently shortening the primary cilium length in several nephron sections [56]. These data confirm that free radical species can modulate the primary cilium length, at least in the kidney, but the mechanism and functional implications of such modulation remain unclear.
