Downregulation of miR-503 in Activated Kidney Fibroblasts Disinhibits KCNN4 in an in Vitro Model of Kidney Fibrosis

Background/Aims: Activated fibroblasts are key controllers of extracellular matrix turnover in kidney fibrosis, the pathophysiological end stage of chronic kidney disease. The proliferation of activated fibroblasts depends on the expression of the calcium-dependent potassium channel KCNN4. Expression of this ion channel is upregulated in fibrotic kidneys. Genetic and pharmacological blockade of KCNN4 inhibits fibrosis in vitro and in vivo. Methods: We studied the regulation of KCNN4 and possible involvement of miRNAs in an in-vitro fibrosis model using murine kidney fibroblasts. We tested fibroblast proliferation, channel function, channel expression and expression regulation after FGF-2 stimulation. Results: Proliferation was significantly increased by FGF-2, channel current and expression were almost doubled (+ 91% and +125%, respectively). MiRNA microarray identified upregulation of miRNA-503, which targets RAF1 and thereby controls KCNN4-expression via disinhibition of the Ras/Raf/MEK/ ERK-cascade. Conclusion: This data show a) a profound upregulation of KCNN4 in stimulated fibroblast and b) identifies miR-503 as a regulator of KCNN4 expression

Arterial Response to Shear Stress Critically Depends on Endothelial TRPV4 Expression

BACKGROUND: In blood vessels, the endothelium is a crucial signal transduction interface in control of vascular tone and blood pressure to ensure energy and oxygen supply according to the organs' needs. In response to vasoactive factors and to shear stress elicited by blood flow, the endothelium secretes vasodilating or vasocontracting autacoids, which adjust the contractile state of the smooth muscle. In endothelial sensing of shear stress, the osmo- and mechanosensitive Ca(2+)-permeable TRPV4 channel has been proposed to be candidate mechanosensor. Using TRPV4(-/-) mice, we now investigated whether the absence of endothelial TRPV4 alters shear-stress-induced arterial vasodilation. METHODOLOGY/PRINCIPAL FINDINGS: In TRPV4(-/-) mice, loss of the TRPV4 protein was confirmed by Western blot, immunohistochemistry and by in situ-patch-clamp techniques in carotid artery endothelial cells (CAEC). Endothelium-dependent vasodilation was determined by pressure myography in carotid arteries (CA) from TRPV4(-/-) mice and wild-type littermates (WT). In WT CAEC, TRPV4 currents could be elicited by TRPV4 activators 4alpha-phorbol-12,13-didecanoate (4alphaPDD), arachidonic acid (AA), and by hypotonic cell swelling (HTS). In striking contrast, in TRPV4(-/-) mice, 4alphaPDD did not produce currents and currents elicited by AA and HTS were significantly reduced. 4alphaPDD caused a robust and endothelium-dependent vasodilation in WT mice, again conspicuously absent in TRPV4(-/-) mice. Shear stress-induced vasodilation could readily be evoked in WT, but was completely eliminated in TRPV4(-/-) mice. In addition, flow/reperfusion-induced vasodilation was significantly reduced in TRPV4(-/-) vs. WT mice. Vasodilation in response to acetylcholine, vasoconstriction in response to phenylephrine, and passive mechanical compliance did not differ between genotypes, greatly underscoring the specificity of the above trpv4-dependent phenotype for physiologically relevant shear stress. CONCLUSIONS/SIGNIFICANCE: Genetically encoded loss-of-function of trpv4 results in a loss of shear stress-induced vasodilation, a response pattern critically dependent on endothelial TRPV4 expression. Thus, Ca(2+)-influx through endothelial TRPV4 channels is a molecular mechanism contributing significantly to endothelial mechanotransduction

Modulation of K(Ca)3.1 channels by eicosanoids, omega-3 fatty acids, and molecular determinants.

Cytochrome P450- and ω-hydrolase products (epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraeonic acid (20-HETE)), natural omega-3 fatty acids (ω3), and pentacyclic triterpenes have been proposed to contribute to a wide range of vaso-protective and anti-fibrotic/anti-cancer signaling pathways including the modulation of membrane ion channels. Here we studied the modulation of intermediate-conductance Ca(2+)/calmodulin-regulated K(+) channels (K(Ca)3.1) by EETs, 20-HETE, ω3, and pentacyclic triterpenes and the structural requirements of these fatty acids to exert channel blockade.We studied modulation of cloned human hK(Ca)3.1 and the mutant hK(Ca)3.1(V275A) in HEK-293 cells, of rK(Ca)3.1 in aortic endothelial cells, and of mK(Ca)3.1 in 3T3-fibroblasts by inside-out and whole-cell patch-clamp experiments, respectively. In inside-out patches, Ca(2+)-activated hK(Ca)3.1 were inhibited by the ω3, DHA and α-LA, and the ω6, AA, in the lower µmolar range and with similar potencies. 5,6-EET, 8,9-EET, 5,6-DiHETE, and saturated arachidic acid, had no appreciable effects. In contrast, 14,15-EET, its stable derivative, 14,15-EEZE, and 20-HETE produced channel inhibition. 11,12-EET displayed less inhibitory activity. The K(Ca)3.1(V275A) mutant channel was insensitive to any of the blocking EETs. Non-blocking 5,6-EET antagonized the inhibition caused by AA and augmented cloned hK(Ca)3.1 and rK(Ca)3.1 whole-cell currents. Pentacyclic triterpenes did not modulate K(Ca)3.1 currents.Inhibition of K(Ca)3.1 by EETs (14,15-EET), 20-HETE, and ω3 critically depended on the presence of electron double bonds and hydrophobicity within the 10 carbons preceding the carboxyl-head of the molecules. From the physiological perspective, metabolism of AA to non-blocking 5,6,- and 8,9-EET may cause AA-de-blockade and contribute to cellular signal transduction processes influenced by these fatty acids

Moderate antagonism of AA-mediated hK_Ca3.1-inhibtion by 5,6-EET.

<p>A) Time course of channel inhibition by 10 µM of AA in the presence of 1 µM 5,6-EET. B) Summary data of channel inhibition at 20 s after seal excision and with two concentrations (1 and 10 µM) of 5,6-EET and AA. Data are means ± SEM; numbers in the graphs indicate the number of inside-out experiments; *<i>P</i><0.05 vs. AA alone, One-way ANOVA and Tukey <i>post hoc</i> test.</p

Membrane expression of cloned human K_Ca3.1 in HEK-293 in inside-out patches and basic pharmacological characterization.

<p>A) From left to right: Exemplary traces of immediate activation of hK_Ca3.1-outward currents upon excision of the patch into 3 µM Ca²⁺-containing bath solution (as indicated by arrow). K_Ca-outward currents are absent in non-transfected HEK-293. Inhibition of hK_Ca3.1-outward currents by charybdotoxin (100 nM, in the pipette solution) and TRAM-34 (1 µM, in the bath solution). B) Inhibition of hK_Ca3.1 by ω3 and arachidonic acid. From left to right: Time course of inactivation of hK_Ca3.1 by docosahexaenoic acid (DHA, 10 µM), arachidonic acid (AA, 10 µM), α-linolenic acid (α-LA, µM) over time. Saturated arachidic acid (ArA, 10 µM) did not affect channel activity. C) Concentration-dependence of inhibition. Note that half of the current was inhibited by AA, DHA, and α-LA at approx. 1 µM. D) Time course of channel inactivation by two concentrations of AA, DHA, and α-LA over time. Data are means ± SEM (% inhibition of K_Ca3.1-current normalized to initial peak amplitude after patch-excision); numbers in the graphs indicate the number of inside-out experiments; *<i>P</i><0.05 vs. vehicle (Ve); One-way ANOVA and Tukey <i>post hoc</i> test.</p

5,6-EET-potentiation of K_Ca3.1 currents.

<p>A) Whole-cell current traces; from left to right: potentiation of Ca²⁺-pre-activated hK_Ca3.1 by 5,6-EET (1 µM) followed by inhibition of the current by AA (10 µM), insensitivity of the hK_Ca3.1^T250S/V275A mutant to 5,6-EET, and insensitivity of the hK_Ca3.1^T250S/V275A mutant to AA (10 µM) and TRAM-34 (1 µM). The hK_Ca3.1 currents were pre-activated by 250 nM Ca²⁺. Panel on the right: summary data. B) From left to right: Ca²⁺-pre-activation of rat endothelial rK_Ca3.1 by 3 µM Ca²⁺ and current inhibition by 14,15-EET (1 µM), larger currents in the presence of 5,6-EET (1 µM) and inhibition by AA (10 µM). Panel on right: Summary data: dependence of 5,6-EET-potentiation on the intracellular Ca²⁺. Note that at a low intracellular Ca²⁺ (0.1 µM) that is below/near the threshold for K_Ca3.1 activation, 5,6-EET did not potentiate the current. In contrast, potentiation occurred at an intracellular Ca²⁺ concentration that is near the EC₅₀ for Ca²⁺-activation of K_Ca3.1 as well as at a saturating Ca²⁺ concentration. C) DHA (1 µM) blocked Ca²⁺-pre-activated mK_Ca3.1 in murine fibroblasts. D) Pentacyclic triterpenes did not modulate murine fibroblast mK_Ca3.1 at a concentration of 1 µM. Data are means ± SEM (% inhibition of K_Ca3.1-current normalized to initial peak amplitude after establishing electrical access (by seal rupture) and stable Ca²⁺-activation of K_Ca3.1-outward currents); Numbers in the graphs indicate the number of whole-cell experiments; *<i>P</i><0.05 vs. control (peak amplitude of the K_Ca3.1-current in the respective cell); One-way ANOVA and Tukey <i>post hoc</i> test.</p

Insensitivity of hK_Ca3.1 mutants.

<p>A) Representative current traces obtained from inside-out recordings using HEK-293 expressing the hK_Ca3.1^V275A mutant. B) Summary data from experiments using the three different hK_Ca3.1 mutants and wt hK_Ca3.1. Concentration of all compounds was 10 µM. Data are means ± SEM; numbers in the graphs indicate the number of inside-out experiments. *<i>P</i><0.05 vs. wt; One-way ANOVA and Tukey <i>post hoc</i> test.</p

Chemical structures of eicosanoids, ω3, and pentacyclic triterpenes and schematic overview of blocking efficacy (decreasing from top to bottom) or non-blocking efficacy.

<p>Chemical structures of eicosanoids, ω3, and pentacyclic triterpenes and schematic overview of blocking efficacy (decreasing from top to bottom) or non-blocking efficacy.</p