Knockout of mitochondrial voltage-dependent anion channel type 3 increases reactive oxygen species (ROS) levels and alters renal sodium transport.

It has been suggested that voltage-dependent anion channels (VDACs) control the release of superoxide from mitochondria. We have previously shown that reactive oxygen species (ROS) such as superoxide (O2̇̄) and hydrogen peroxide (H2O2) stimulate epithelial sodium channels (ENaCs) in sodium-transporting epithelial tissue, including cortical collecting duct (CCD) principal cells. Therefore, we hypothesized that VDACs could regulate ENaC by modulating cytosolic ROS levels. Herein, we find that VDAC3-knockout(KO) mice can maintain normal salt and water balance on low-salt and high-salt diets. However, on a high-salt diet for 2 weeks, VDAC3-KO mice had significantly higher systolic blood pressure than wildtype mice. Consistent with this observation, after a high-salt diet for 2 weeks, ENaC activity in VDAC3-KO mice was significantly higher than wildtype mice. EM analysis disclosed a significant morphological change of mitochondria in the CCD cells of VDAC3-KO mice compared with wildtype mice, which may have been caused by mitochondrial superoxide overload. Of note, compared with wildtype animals, ROS levels in VDAC3-KO animals fed a normal or high-salt diet were consistently and significantly increased in renal tubules. Both the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL) and the mitochondrial ROS scavenger (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mito-TEMPO) could reverse the effect of high-salt on ENaC activity and systolic blood pressure in the VDAC3-KO mice. Mito-TEMPO partially correct the morphological changes in VDAC3-KO mice. Our results suggest that knocking out mitochondrial VDAC3 increases ROS, alters renal sodium transport, and leads to hypertension.

ENaC activity is regulated by calpain-2 proteolysis of MARCKS proteins.

We previously demonstrated a role for the myristoylated alanine-rich C kinase substrate (MARCKS) to serve as an adaptor protein in the anionic phospholipid phosphate-dependent regulation of the epithelial sodium channel (ENaC). Both MARCKS and ENaC are regulated by proteolysis. Calpains are a family of ubiquitously expressed intracellular Ca2+-dependent cysteine proteases involved in signal transduction. Here we examine the role of calpain-2 in regulating MARCKS and ENaC in cultured renal epithelial cells and in the mouse kidney. Using recombinant fusion proteins, we show that MARCKS, but not the ENaC subunits, are a substrate of calpain-2 in the presence of Ca2+ Pharmacological inhibition of calpain-2 alters MARCKS protein expression in light-density sucrose gradient fractions from cell lysates of mouse cortical collecting duct cells. Calpain-dependent cleaved products of MARCKS are detectable in cultured renal cells. Ca2+ mobilization and calpain-2 inhibition decrease the association between ENaC and MARCKS. The inhibition of calpain-2 reduces ENaC activity as demonstrated by single-channel patch-clamp recordings and transepithelial current measurements. These results suggest that calpain-2 proteolysis of MARCKS promotes its interaction with lipids and ENaC at the plasma membrane to allow for the phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent regulation of ENaC activity in the kidney.

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit.

Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the ß and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.

Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC.

A thin fluid layer in alveoli is normal and results from a balance of fluid entry and fluid uptake by transepithelial salt and water reabsorption. Conventional wisdom suggests the reabsorption is via epithelial Na+ channels (ENaC), but if all Na+ reabsorption were via ENaC, then amiloride, an ENaC inhibitor, should block alveolar fluid clearance (AFC). However, amiloride blocks only half of AFC. The reason for failure to block is clear from single-channel measurements from alveolar epithelial cells: ENaC channels are observed, but another channel is present at the same frequency that is nonselective for Na+ over K+, has a larger conductance, and has shorter open and closed times. These two channel types are known as highly selective channels (HSC) and nonselective cation channels (NSC). HSC channels are made up of three ENaC subunits since knocking down any of the subunits reduces HSC number. NSC channels contain α-ENaC since knocking down α-ENaC reduces the number of NSC (knocking down ß- or γ-ENaC has no effect on NSC, but the molecular composition of NSC channels remains unclear). We show that NSC channels consist of at least one α-ENaC and one or more acid-sensing ion channel 1a (ASIC1a) proteins. Knocking down either α-ENaC or ASIC1a reduces both NSC and HSC number, and no NSC channels are observable in single-channel patches on lung slices from ASIC1a knockout mice. AFC is reduced in knockout mice, and wet wt-to-dry wt ratio is increased, but the percentage increase in wet wt-to-dry wt ratio is larger than expected based on the reduction in AFC.

The Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria.

The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca(2+)]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca(2+)]i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca(2+)]i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca(2+)]i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca(2+)]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca(2+)]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca(2+)]i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca(2+)]i, creating [Ca(2+)]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca(2+)]i uptake destroyed the polarized response of ENaC to [Ca(2+)]i. Overall, our data suggest that ENaC is regulated by [Ca(2+)]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca(2+)]i sequestration.

Acute ethanol induces apoptosis by stimulating TRPC6 via elevation of superoxide in oxygenated podocytes.

Our recent studies indicate that hydrogen peroxide (H2O2) only at high concentrations can cause oxidative stress in renal epithelial cells and induce apoptosis of podocytes. Consistently, the present study shows that H2O2, even at 1 mM, failed to induce intracellular oxidative stress and apoptosis of the podocytes due to efficient activity of catalase, an enzyme which degrades H2O2 to produce water and oxygen (O2). However, H2O2 acted as a source of O2 to allow acute ethanol to induce superoxide production and cause apoptosis of the podocytes. In contrast, acute ethanol alone did not elevate intracellular superoxide, even though it stimulates expression and translocation of p47phox to the plasma membrane. Inhibition of catalase abolished not only O2 production from H2O2 degradation, but also NOX2-dependent superoxide production in the podocytes challenged by both H2O2 and acute ethanol. In parallel, acute ethanol in the presence of H2O2, but neither ethanol nor H2O2 alone, stimulated transient receptor potential canonical 6 (TRPC6) channels and caused TRPC6-dependent elevation of intracellular Ca2+. These data suggest that exogenous H2O2 does not induce oxidative stress due to rapid degradation to produce O2 in the podocytes, but the oxygenated podocytes become sensitive to acute ethanol challenge and undergo apoptosis via a TRPC6-dependent elevation of intracellular Ca2+. Since cultured podocytes are considered in hypoxic conditions, H2O2 may be used as a source of O2 to establish an ischemia-reperfusion model in some type of cultured cells in which H2O2 does not directly induce intracellular oxidative stress.

Hydrogen peroxide suppresses TRPM4 trafficking to the apical membrane in mouse cortical collecting duct principal cells.

A Ca2+-activated nonselective cation channel (NSCCa) is found in principal cells of the mouse cortical collecting duct (CCD). However, the molecular identity of this channel remains unclear. We used mpkCCDc14 cells, a mouse CCD principal cell line, to determine whether NSCCa represents the transient receptor potential (TRP) channel, the melastatin subfamily 4 (TRPM4). A Ca2+-sensitive single-channel current was observed in inside-out patches excised from the apical membrane of mpkCCDc14 cells. Like TRPM4 channels found in other cell types, this channel has an equal permeability for Na+ and K+ and has a linear current-voltage relationship with a slope conductance of ~23 pS. The channel was inhibited by a specific TRPM4 inhibitor, 9-phenanthrol. Moreover, the frequency of observing this channel was dramatically decreased in TRPM4 knockdown mpkCCDc14 cells. Unlike those previously reported in other cell types, the TRPM4 in mpkCCDc14 cells was unable to be activated by hydrogen peroxide (H2O2). Conversely, after treatment with H2O2, TRPM4 density in the apical membrane of mpkCCDc14 cells was significantly decreased. The channel in intact cell-attached patches was activated by ionomycin (a Ca2+ ionophore), but not by ATP (a purinergic P2 receptor agonist). These data suggest that the NSCCa current previously described in CCD principal cells is actually carried through TRPM4 channels. However, the physiological role of this channel in the CCD remains to be further determined.

Lovastatin inhibits human B lymphoma cell proliferation by reducing intracellular ROS and TRPC6 expression.

Clinical evidence suggests that statins reduce cancer incidence and mortality. However, there is lack of in vitro data to show the mechanism by which statins can reduce the malignancies of cancer cells. We used a human B lymphoma Daudi cells as a model and found that lovastatin inhibited, whereas exogenous cholesterol (Cho) stimulated, proliferation cell cycle progression in control Daudi cells, but not in the cells when transient receptor potential canonical 6 (TRPC6) channel was knocked down. Lovastatin decreased, whereas Cho increased, the levels of intracellular reactive oxygen species (ROS) respectively by decreasing or increasing the expression of p47-phox and gp91-phox (NOX2). Reducing intracellular ROS with either a mimetic superoxide dismutase (TEMPOL) or an NADPH oxidase inhibitor (apocynin) inhibited cell proliferation, particularly in Cho-treated cells. The effects of TEMPOL or apocynin were mimicked by inhibition of TRPC6 with SKF-96365. Lovastatin decreased TRPC6 expression and activity via a Cho-dependent mechanism, whereas Cho increased TRPC6 expression and activity via an ROS-dependent mechanism. Consistent with the fact that TRPC6 is a Ca(2+)-permeable channel, lovastatin decreased, but Cho increased, intracellular Ca(2+) also via ROS. These data suggest that lovastatin inhibits malignant B cell proliferation by reducing membrane Cho, intracellular ROS, TRPC6 expression and activity, and intracellular Ca(2+).

ENaC activity is increased in isolated, split-open cortical collecting ducts from protein kinase Cα knockout mice.

The epithelial Na channel (ENaC) is negatively regulated by protein kinase C (PKC) as shown using PKC activators in a cell culture model. To determine whether PKCα influences ENaC activity in vivo, we examined the regulation of ENaC in renal tubules from PKCα⁻/⁻ mice. Cortical collecting ducts were dissected and split open, and the exposed principal cells were subjected to cell-attached patch clamp. In the absence of PKCα, the open probability (P0) of ENaC was increased three-fold vs. wild-type SV129 mice (0.52 ± 0.04 vs. 0.17 ± 0.02). The number of channels per patch was also increased. Using confocal microscopy, we observed an increase in membrane localization of α-, ß-, and γ-subunits of ENaC in principal cells in the cortical collecting ducts of PKCα⁻/⁻ mice compared with wild-type mice. To confirm this increase, one kidney from each animal was perfused with biotin, and membrane protein was pulled down with streptavidin. The nonbiotinylated kidney was used to assess total protein. While total ENaC protein did not change in PKCα⁻/⁻ mice, membrane localization of all the ENaC subunits was increased. The increase in membrane ENaC could be explained by the observation that ERK1/2 phosphorylation was decreased in the knockout mice. These results imply a reduction in ENaC membrane accumulation and P0 by PKCα in vivo. The PKC-mediated increase in ENaC activity was associated with an increase in blood pressure in knockout mice fed a high-salt diet.

Basolateral P2X4channels stimulate ENaC activity in Xenopus cortical collecting duct A6 cells.

The polarized nature of epithelial cells allows for different responses to luminal or serosal stimuli. In kidney tubules, ATP is produced luminally in response to changes in luminal flow. Luminal increases in ATP have been previously shown to inhibit the renal epithelial Na⁺ channel (ENaC). On the other hand, ATP is increased basolaterally in renal epithelia in response to aldosterone. We tested the hypothesis that basolateral ATP can stimulate ENaC function through activation of the P2X4receptor/channel. Using single channel cell-attached patch-clamp techniques, we demonstrated the existence of a basolaterally expressed channel stimulated by the P2X4agonist 2-methylthio-ATP (meSATP) in Xenopus A6 cells, a renal collecting duct principal cell line. This channel had a similar reversal potential and conductance to that of P2X4channels. Cell surface biotinylation of the basolateral side of these cells confirmed the basolateral presence of the P2X4 receptor. Basolateral addition of meSATP enhanced the activity of ENaC in single channel patch-clamp experiments, an effect that was absent in cells transfected with a dominant negative P2X4receptor construct, indicating that activation of P2X4channels stimulates ENaC activity in these cells. The effect of meSATP on ENaC activity was reduced after chelation of basolateral Ca²âº with EGTA or inhibition of phosphatidylinositol 3-kinase with LY-294002. Overall, our results show that ENaC is stimulated by P2X4receptor activation and that the stimulation is dependent on increases in intracellular Ca²âº and phosphatidylinositol 3-kinase activation.

Aldosterone acutely stimulates NCC activity via a SPAK-mediated pathway.

Hypertension is a leading cause of morbidity and mortality worldwide, and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 h) of aldosterone on NCC in the rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. This effect was dependent upon the presence of the mineralocorticoid receptor and serum- and glucocorticoid-regulated kinase 1 (SGK1). Furthermore, STE20/SPS-1-related proline/alanine-rich kinase (SPAK) phosphorylation also increased, and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via a minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity.

Lack of protein kinase C-α leads to impaired urine concentrating ability and decreased aquaporin-2 in angiotensin II-induced hypertension.

Regulation of water and urea transport in the inner medullary collecting duct is essential for urine concentration. Aquaporin (AQP)2 water channels and urea transporter (UT)-A1 are inserted into the apical membrane upon phosphorylation of the channels to allow the transcellular movement of water and urea. Since ANG II activates PKC in many cell types, we tested the hypothesis that ANG II-induced regulation of water and urea transport is mediated by PKC. Osmotic minipumps delivered ANG II to wild-type (WT) or PKC-α(-/-) mice for 7 days. Inner medullas were harvested, and protein abundance was determined by immunoblot. ANG II increased systolic blood pressure to a similar degree in WT and PKC-α(-/-) mice. ANG II had no effect on the urine output of WT mice but increased that of PKC-α(-/-) mice. In accordance with observed differences in urine output, AQP2 abundance was unchanged in ANG II-treated WT animals but was decreased in PKC-α(-/-) mice. No change in membrane accumulation was seen. Phosphorylation of the cAMP-induced transcription factor CREB was decreased in PKC-α(-/-) mice in response to ANG II with no change in overall CREB abundance. ANG II did not alter the abundance of UT-A1 protein in WT or PKC-α(-/-) mice. Phosphorylation and overall abundance of tonicity-responsive enhancer-binding protein, a transcription factor that regulates UT-A1, were also unaltered by ANG II in either group. We conclude that PKC-α protects against ANG II-induced decreases in urine concentrating ability by maintaining AQP2 levels through CREB phosphorylation.

N-methyl-D-aspartate receptor subunit NR3a expression and function in principal cells of the collecting duct.

N-methyl-D-aspartate receptors (NMDARs) are Ca(2+)-permeable, ligand-gated, nonselective cation channels that function as neuronal synaptic receptors but which are also expressed in multiple peripheral tissues. Here, we show for the first time that NMDAR subunits NR3a and NR3b are highly expressed in the neonatal kidney and that there is continued expression of NR3a in the renal medulla and papilla of the adult mouse. NR3a was also expressed in mIMCD-3 cells, where it was found that hypoxia and hypertonicity upregulated NR3a expression. Using short-hairpin (sh) RNA-based knockdown, a stable inner medullary collecting duct (IMCD) cell line was established that had ∼80% decrease in NR3a. Knockdown cells exhibited an increased basal intracellular calcium concentration, reduced cell proliferation, and increased cell death. In addition, NR3a knockdown cells exhibited reduced water transport in response to the addition of vasopressin, suggesting an alteration in aquaporin-2 (AQP2) expression/function. Consistent with this notion, we demonstrate decreased surface expression of glycosylated AQP2 in IMCD cells transfected with NR3a shRNA. To determine whether this also occurred in vivo, we compared AQP2 levels in wild-type vs. in NR3a(-/-) mice. Total AQP2 protein levels in the outer and inner medulla were significantly reduced in knockout mice compared with control mice. Finally, NR3a(-/-) mice showed a significant delay in their ability to increase urine osmolality during water restriction. Thus NR3a may play a renoprotective role in collecting duct cells. Therefore, under conditions that are associated with high vasopressin levels, NR3a, by maintaining low intracellular calcium levels, protects the function of the principal cells to reabsorb water and thereby increase medullary osmolality.

Mice lacking the ADP ribosyl cyclase CD38 exhibit attenuated renal vasoconstriction to angiotensin II, endothelin-1, and norepinephrine.

ADP ribosyl (ADPR) cyclases comprise a family of ectoenzymes recently shown to influence cytosolic Ca(2+) concentration in a variety of cell types. At least two ADPR cyclase family members have been identified in mammals: CD38 and CD157. We recently found reduced renal vascular reactivity to angiotensin II (ANG II), endothelin-1 (ET-1), and norepinephrine (NE) in the presence of the broad ADPR cyclase inhibitor nicotinamide. We hypothesized that CD38 mediates effects attributed to ADPR cyclase. We found expression of ADPR cyclases CD38 and CD157 mRNA in spleen, thymus, skin, and preglomerular arterioles of wild-type (WT) animals. Mice lacking CD38 showed decreased CD157 expression in most tissues tested. No difference in systolic or mean arterial pressure was observed between strains in either conscious or anesthetized states, whereas heart rate was reduced 10-20% in CD38-/- animals (P < 0.05). During anesthesia, CD38-/- mice had reduced basal renal blood flow (RBF) and urine excretion (P < 0.05). RBF responses to intravenous injection of ANG II, ET-1, and NE were attenuated approximately 50% in CD38-/- vs. WT mice (P < 0.01 for all). The systemic pressor response to ANG II was decreased in the absence of CD38 (P < 0.01), whereas that to NE was normal (P > 0.05); ET-1 was administered at a nonpressor dose. Nicotinamide effectively inhibited ANG II-induced renal vasoconstriction in WT mice (P < 0.001), but had no effect on renal responses to ANG II in CD38-/- mice (P > 0.5). Overall, our observations indicate the presence of two ADPR cyclase family members in renal preglomerular resistance arterioles and the importance of CD38 participation in acute vascular responses to all three vasoconstrictors in the renal microcirculation.

NAADP receptors mediate calcium signaling stimulated by endothelin-1 and norepinephrine in renal afferent arterioles.

The enzyme ADP-ribosyl (ADPR) cyclase plays a significant role in mediating increases in renal afferent arteriolar cytosolic calcium concentration ([Ca(2+)](i)) in vitro and renal vasoconstriction in vivo. ADPR cyclase produces cyclic ADP ribose, a second messenger that contributes importantly to ryanodine receptor-mediated Ca(2+) mobilization in renal vascular responses to several vasoconstrictors. Recent studies in nonrenal vascular smooth muscle cells (VSMC) have shown that nicotinic acid adenine dinucleotide phosphate (NAADP), another second messenger generated by ADPR cyclase, may contribute to Ca(2+) signaling. We tested the hypothesis that a Ca(2+) signaling pathway involving NAADP receptors participates in afferent arteriolar [Ca(2+)](i) responses to the G protein-coupled receptor agonists endothelin-1 (ET-1) and norepinephrine (NE). To test this, we isolated rat renal afferent arterioles and measured [Ca(2+)](I) using fura-2 fluorescence. We compared peak [Ca(2+)](i) increases stimulated by ET-1 and NE in the presence and absence of inhibitors of acidic organelle-dependent Ca(2+) signaling and NAADP receptors. Vacuolar H(+)-ATPase inhibitors bafilomycin A1 and concanamycin A, disruptors of pH and Ca(2+) stores of lysosomes and other acidic organelles, individually antagonized [Ca(2+)](i) responses to ET-1 and NE by 40-50% (P < 0.05). The recently discovered NAADP receptor inhibitor Ned-19 attenuated [Ca(2+)](i) responses to ET-1 or NE by 60-70% (P < 0.05). We conclude that NAADP receptors contribute to both ET-1- and NE-induced [Ca(2+)](i) responses in afferent arterioles, an effect likely dependent on acidic vesicle, possibly involving lysosome, signaling in VSMC in the renal microcirculation.

Regulation of the renal microcirculation by ryanodine receptors and calcium-induced calcium release.

PURPOSE OF REVIEW: Emerging evidence highlights the importance of physiological participation of ryanodine receptors (RyR) and Ca-induced-Ca-release (CICR) from the sarcoplasmic reticulum in Ca signaling and arteriolar contraction in the renal microcirculation. RECENT FINDINGS: Adenosine diphosphate -ribosyl (ADPR) cyclase and its endogenous metabolites cyclic adenosine diphosphate-ribose and nicotinic acid adenine dinucleotide phosphate mobilize intracellular Ca from sarcoplasmic reticulum stores in the renal vasculature via actions on RyR. The ADPR cyclase/cyclic adenosine diphosphate-ribose/RyR/CICR second messenger system mediates significant (>50%) changes in cytosolic Ca concentration ([Ca]i) and contractile function of preglomerular arteries/arterioles during angiotensin II and endothelin-1 stimulation of G-protein coupled receptors. These receptors rapidly activate ADPR cyclase via stimulation of superoxide (O2) production by nicotinamide adenine dinucleotide phosphate oxidases. Basal ADPR cyclase activity and RyR/CICR contribute to [Ca]i responses initiated by Ca entry and by inositol trisphosphate receptor-induced sarcoplasmic reticulum Ca release. Acute [Ca]i responses in isolated afferent arterioles and renal vasoconstriction in vivo are attenuated by more than 50% by pharmacological inhibition of ADPR cyclase or RyR. Similarly, renal vascular reactivity to angiotensin II, endothelin-1 and norepinephrine is attenuated by approximately 50% in mice lacking CD38, the main mammalian ADPR cyclase. CONCLUSION: RyR and CICR are important regulations of Ca signaling and contractile tone of renal resistance arterioles in healthy kidneys. The role of this novel-signaling pathway in pathophysiological mechanisms awaits investigation.

Lovastatin attenuates hypertension induced by renal tubule-specific knockout of ATP-binding cassette transporter A1, by inhibiting epithelial sodium channels.

BACKGROUND AND PURPOSE: We have shown that cholesterol is synthesized in the principal cells of renal cortical collecting ducts (CCD) and stimulates the epithelial sodium channels (ENaC). Here we have determined whether lovastatin, a cholesterol synthesis inhibitor, can antagonize the hypertension induced by activated ENaC, following deletion of the cholesterol transporter (ATP-binding cassette transporter A1; ABCA1). EXPERIMENTAL APPROACH: We selectively deleted ABCA1 in the principal cells of mouse CCD and used the cell-attached patch-clamp technique to record ENaC activity. Western blot and immunofluorescence staining were used to evaluate protein expression levels. Systolic BP was measured with the tail-cuff method. KEY RESULTS: Specific deletion of ABCA1 elevated BP and ENaC single-channel activity in the principal cells of CCD in mice. These effects were antagonized by lovastatin. ABCA1 deletion elevated intracellular cholesterol levels, which was accompanied by elevated ROS, increased expression of serum/glucocorticoid regulated kinase 1 (Sgk1), phosphorylated neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) and furin, along with shorten the primary cilium, and reduced ATP levels in urine. CONCLUSIONS AND IMPLICATIONS: These data suggest that specific deletion of ABCA1 in principal cells increases BP by stimulating ENaC channels via a cholesterol-dependent pathway which induces several secondary responses associated with oxidative stress, activated Sgk1/Nedd4-2, increased furin expression, and reduced cilium-mediated release of ATP. As ABCA1 can be blocked by cyclosporine A, these results suggest further investigation of the possible use of statins to treat CsA-induced hypertension.

Inhibition of TRPC6 reduces non-small cell lung cancer cell proliferation and invasion.

Recent studies indicate that the transient receptor potential canonical 6 (TRPC6) channel is highly expressed in several types of cancer cells. However, it remains unclear whether TRPC6 contributes to the malignancy of human non-small cell lung cancer (NSCLC). We used a human NSCLC A549 cell line as a model and found that pharmacological blockade or molecular knockdown of TRPC6 channel inhibited A549 cell proliferation by arresting cell cycle at the S-G2M phase and caused a significant portion of cells detached and rounded-up, but did not induce any types of cell death. Western blot and cell cycle analysis show that the detached round cells at the S-G2M phase expressed more TRPC6 than the still attached polygon cells at the G1 phase. Patch-clamp data also show that TRPC whole-cell currents in the detached cells were significantly higher than in the still attached cells. Inhibition of Ca2+-permeable TRPC6 channels significantly reduced intracellular Ca2+ in A549 cells. Interestingly, either blockade or knockdown of TRPC6 strongly reduced the invasion of this NSCLC cell line and decreased the expression of an adherent protein, fibronectin, and a tight junction protein, zonula occluden protein-1 (ZO-1). These data suggest that TRPC6-mediated elevation of intracellular Ca2+ stimulates NSCLC cell proliferation by promoting cell cycle progression and that inhibition of TRPC6 attenuates cell proliferation and invasion. Therefore, further in vivo studies may lead to a consideration of using a specific TRPC6 blocker as a complement to treat NSCLC.

ADP-ribosyl cyclase and ryanodine receptors mediate endothelin ETA and ETB receptor-induced renal vasoconstriction in vivo.

ADP-ribosyl cyclase (ADPR cyclase) and ryanodine receptors (RyR) participate in calcium transduction in isolated afferent arterioles. We hypothesized that this signaling pathway is activated by ETA and ETB receptors in the renal vasculature to mediate vasoconstriction in vivo. To test this, we measured acute renal blood flow (RBF) responses to ET-1 in anesthetized rats and mice in the presence and absence of functional ADPR cyclase and/or RyR. Inhibitors of ADPR cyclase (nicotinamide) or RyR (ruthenium red) reduced RBF responses to ET-1 by 44% (P < 0.04 for both) in Sprague-Dawley rats. Mice lacking the predominant form of ADPR cyclase (CD38-/-) had RBF responses to ET-1 that were 47% weaker than those seen in wild-type mice (P = 0.01). Selective ETA receptor stimulation (ET-1+BQ788) produced decreases in RBF that were attenuated by 43 and 56% by nicotinamide or ruthenium red, respectively (P < 0.02 for both). ADPR cyclase or RyR inhibition also reduced vasoconstrictor effects of the ETB receptor agonist sarafotoxin 6c (S6c; 77 and 54%, respectively, P < 0.02 for both). ETB receptor stimulation by ET-1 + the ETA receptor antagonist BQ123 elicited responses that were attenuated by 59 and 60% by nicotinamide and ruthenium red, respectively (P < 0.01 for both). Nicotinamide attenuated RBF responses to S6c by 54% during inhibition of nitric oxide synthesis (P = 0.001). We conclude that in the renal microcirculation in vivo 1) ET-1-induced vasoconstriction is mediated by ADPR cyclase and RyR; 2) both ETA and ETB receptors activate this pathway; and 3) ADPR cyclase participates in ETB receptor signaling independently of NO.

ADP-ribosyl cyclase and ryanodine receptor activity contribute to basal renal vasomotor tone and agonist-induced renal vasoconstriction in vivo.

An important role for the enzyme ADP-ribosyl cyclase (ADPR cyclase) and its downstream targets, the ryanodine receptors (RyR), is emerging for a variety of vascular systems. We hypothesized that the ADPR cyclase/RyR pathway contributes to regulation of renal vasomotor tone in vivo. To test this, we continuously measured renal blood flow (RBF) in anesthetized Sprague-Dawley rats. Infusion of the ADPR cyclase inhibitor nicotinamide intrarenally at low doses inhibits angiotensin II (ANG II)- and norepinephrine (NE)-induced vasoconstriction by 72 and 67%, respectively (P < 0.001). RBF studies in rats were extended to mice lacking the predominant form of ADPR cyclase (CD38). Acute renal vasoconstrictor responses to ANG II and NE are impaired by 59 and 52%, respectively, in anesthetized CD38-/- mice compared with wild-type controls (P < 0.05). Intrarenal injection of the RyR activator FK506 decreases RBF by 22% (P > 0.03). Furthermore, RyR inhibition with ruthenium red attenuates ANG II and NE responses by 50 and 59%, respectively (P < or = 0.01). Given at higher doses, nicotinamide increases basal RBF by 22% (P > 0.001). Non-receptor-mediated renal vasoconstriction by L-type voltage-gated Ca(2+) channels is also dependent on ADPR cyclase and RyRs. Nicotinamide and ruthenium red inhibit constriction by the L-type channel agonist BAY K 8644 by 59% (P > 0.02) and 63% (P > 0.001). We conclude that 1) ADPR cyclase activity contributes to regulation of renal vasomotor tone under resting conditions, 2) renal vasoconstriction induced by G protein-coupled receptor agonists ANG II and NE is mediated in part by ADPR cyclase and RyRs, and 3) ADPR cyclase and RyRs participate in L-type channel-mediated renal vasoconstriction in vivo.