4. The regulation of ciliary function

C-terminus is involved in intracellular signaling and interaction with PKD2 [34, 35]. PKD1 has

PKD2, a 968 amino acids long protein, is a non-selective Ca2+ permeable transient receptor potential (TRP) channel consisting of six membrane-spanning domains and intracellular Cand N-terminal domains [37]. The sensory function of PKD2 depends on PKD1 and has to be localized to endothelial primary cilia [38]. Accordingly, PKD2 functioning as a Ca2+ channel [29] allows extracellular Ca2+ influx into the cilioplasm in response to FSS [39]. Thus, mechanistically, PKD1 and PKD2 interact through their C-terminus [29, 34, 35] and localized to the ciliary membrane; they are able to detect extracellular FSS and to increase cytosolic Ca2+. This

A series of mutation and deletion experiments demonstrated that besides PKD1 and PKD2, the protein polaris also orchestrates FSS sensing. The physiological Ca2+ and NO increase in response to FSS is abolished when the pkd1, pkd2 and polaris genes are mutated or knocked out [29]. Interestingly, mutations or deletion of polaris seem to affect the structural integrity of cilia through the PKD1 and PKD2 mislocalization, which remain concentrated at the basal body [9, 29, 32, 41]. Together these findings evidence that polaris mediates the PKD1 and PKD2 primary cilium localization, implying a polaris cilium sensory function regulation. In order to achieve a proper fluid-shear sensing by endothelial cells and an adequate response, all

been shown to mediate fluid-shear sensing in epithelial and endothelial cells [9, 36].

94 Endothelial Dysfunction - Old Concepts and New Challenges

turns on a signaling cascade leading to the production of NO [9, 38, 40].

three components, PKD1, PKD2 and polaris, are thus indispensable.

tion [2, 29]. This particular pathway is summarized in Figure 2.

primary cilium-mediated FSS signaling [29] (Figure 2).

Ca2+ chelator EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N<sup>0</sup>

3.3. Molecular cascade involved in shear stress-induced calcium and NO signaling

FSS leads to cilia bending leading to PKD2-mediated increase of intracellular Ca2+ that leads to activation of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptor (InsP3R) present in the endoplasmic reticulum, which then releases its stores of Ca2+ enhancing the intracellular levels of Ca2+ [42, 43]. Subsequently, Ca2+ activates several intracellular signaling pathways, including the activation of the eNOS-bound calmodulin, thus increasing the production of NO that diffuses from endothelial cells to neighboring VSMC inducing vasodilata-

The works of AbouAlaiwi et al. [29] have helped to elucidate this last mechanism. In order to prove that FSS-dependent primary cilia bending induces extracellular Ca2+ influx, they used

these experiments, EGTA abolished both Ca2+ and NO increases. In addition, the inhibitor of the eNOS, NG-nitro-L-arginine methyl ester (L-NAME) blocked the FSS-induced NO release without affecting Ca2+ increase. The same effect was shown after blocking calcium-dependent mechanisms of NO production using calphostin C as an inhibitor of protein kinase C (PKC) or W7 as antagonist of calmodulin. Similarly, inhibiting protein kinase B (PKB)/Akt abolished NO release without altering Ca2+ increase. Inhibiting IP3 kinase using LY-294002 did not alter neither Ca2+ nor NO increase. These findings indicate that calmodulin, PKC and Akt/PKB are downstream of the calcium pathway and that they are necessary for NO release during

,N<sup>0</sup>


Changes in fluid patterns generate differential biomechanical forces, which lead to alteration of cilia function or structure [2]. Indeed, almost all blood vessels possess cilia [4, 23]. Particularly, arteries with low FSS or high fluid turbulence have cilia [2]. A prolonged exposure of endothelial cells to high FSS induces the disassembly of cilia [28] and inactivation of PKD1 by proteolytic cleavage [9], suggesting that primary cilia may not be required only to sense high shear stress [2].

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 increase of acetylation of MT [18, 44].

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

Sensing Fluid-Shear Stress in the Endothelial System with a Special Emphasis on the Primary Cilium

http://dx.doi.org/10.5772/intechopen.73134

97

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

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.

VEGFs are a complex family of glycoproteins that are structurally related to platelet-derived growth factor (PDGF) [57]. Through alternative RNA splicing, VEGF family is constituted by

6. Vascular endothelial growth factor and shear stress

, and

mechanosensory organelle for FSS inside the nephron tubule via NO.

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 regulation are still unclear.
