TRPV4 expression in the renal tubule is necessary for maintaining whole body K+ homeostasis.

The Ca2+-permeable transient receptor potential vanilloid type 4 (TRPV4) channel serves as the sensor of tubular flow, thus being well suited to govern mechanosensitive K+ transport in the distal renal tubule. Here, we directly tested whether the TRPV4 function is significant in affecting K+ balance. We used balance metabolic cage experiments and systemic measurements with different K+ feeding regimens [high (5% K+), regular (0.9% K+), and low (<0.01% K+)] in newly created transgenic mice with selective TRPV4 deletion in the renal tubule (TRPV4fl/fl-Pax8Cre) and their littermate controls (TRPV4fl/fl). Deletion was verified by the absence of TRPV4 protein expression and lack of TRPV4-dependent Ca2+ influx. There were no differences in plasma electrolytes, urinary volume, and K+ levels at baseline. In contrast, plasma K+ levels were significantly elevated in TRPV4fl/fl-Pax8Cre mice on high K+ intake. K+-loaded knockout mice exhibited lower urinary K+ levels than TRPV4fl/fl mice, which was accompanied by higher aldosterone levels by day 7. Moreover, TRPV4fl/fl-Pax8Cre mice had more efficient renal K+ conservation and higher plasma K+ levels in the state of dietary K+ deficiency. H+-K+-ATPase levels were significantly increased in TRPV4fl/fl-Pax8Cre mice on a regular diet and especially on a low-K+ diet, pointing to augmented K+ reabsorption in the collecting duct. Consistently, we found a significantly faster intracellular pH recovery after intracellular acidification, as an index of H+-K+-ATPase activity, in split-opened collecting ducts from TRPV4fl/fl-Pax8Cre mice. In summary, our results demonstrate an indispensable prokaliuretic role of TRPV4 in the renal tubule in controlling K+ balance and urinary K+ excretion during variations in dietary K+ intake. NEW & NOTEWORTHY The mechanoactivated transient receptor potential vanilloid type 4 (TRPV4) channel is expressed in distal tubule segments, where it controls flow-dependent K+ transport. Global TRPV4 deficiency causes impaired adaptation to variations in dietary K+ intake. Here, we demonstrate that renal tubule-specific TRPV4 deletion is sufficient to recapitulate the phenotype by causing antikaliuresis and higher plasma K+ levels in both states of K+ load and deficiency.

TRPV4 functional status in cystic cells regulates cystogenesis in autosomal recessive polycystic kidney disease during variations in dietary potassium.

Mechanosensitive TRPV4 channel plays a dominant role in maintaining [Ca2+ ]i homeostasis and flow-sensitive [Ca2+ ]i signaling in the renal tubule. Polycystic kidney disease (PKD) manifests as progressive cyst growth due to cAMP-dependent fluid secretion along with deficient mechanosensitivity and impaired TRPV4 activity. Here, we tested how regulation of renal TRPV4 function by dietary K+ intake modulates the rate of cystogenesis and mechanosensitive [Ca2+ ]i signaling in cystic cells of PCK453 rats, a homologous model of human autosomal recessive PKD (ARPKD). One month treatment with both high KCl (5% K+ ) and KB/C (5% K+ with bicarbonate/citrate) diets significantly increased TRPV4 levels when compared to control (0.9% K+ ). High KCl diet caused an increased TRPV4-dependent Ca2+ influx, and partial restoration of mechanosensitivity in freshly isolated monolayers of cystic cells. Unexpectedly, high KB/C diet induced an opposite effect by reducing TRPV4 activity and worsening [Ca2+ ]i homeostasis. Importantly, high KCl diet decreased cAMP, whereas high KB/C diet further increased cAMP levels in cystic cells (assessed as AQP2 distribution). At the systemic level, high KCl diet fed PCK453 rats had significantly lower kidney-to-bodyweight ratio and reduced cystic area. These beneficial effects were negated by a concomitant administration of an orally active TRPV4 antagonist, GSK2193874, resulting in greater kidney weight, accelerated cystogenesis, and augmented renal injury. High KB/C diet also exacerbated renal manifestations of ARPKD, consistent with deficient TRPV4 activity in cystic cells. Overall, we demonstrate that TRPV4 channel activity negatively regulates cAMP levels in cystic cells thus attenuating (high activity) or accelerating (low activity) ARPKD progression.

Adenosine inhibits the basolateral Cl- ClC-K2/b channel in collecting duct intercalated cells.

Adenosine plays an important role in various aspects of kidney physiology, but the specific targets and mechanisms of actions are not completely understood. The collecting duct has the highest expression of adenosine receptors, particularly adenosine A1 receptors (A1Rs). Interstitial adenosine levels are greatly increased up to a micromolar range in response to dietary salt loading. We have previously shown that the basolateral membrane of principal cells has primarily K+ conductance mediated by Kir4.1/5.1 channels to mediate K+ recycling and to set up a favorable driving force for Na+/K+ exchange (47). Intercalated cells express the Cl- ClC-K2/b channel mediating transcellular Cl- reabsorption. Using patch-clamp electrophysiology in freshly isolated mouse collecting ducts, we found that acute application of adenosine reversely inhibits ClC-K2/b open probability from 0.31 ± 0.04 to 0.17 ± 0.06 and to 0.10 ± 0.05 for 1 and 10 µM, respectively. In contrast, adenosine (10 µM) had no measureable effect on Kir4.1/5.1 channel activity in principal cells. The inhibitory effect of adenosine on ClC-K2/b was abolished in the presence of the A1R blocker 8-cyclopentyl-1,3-dipropylxanthine (10 µM). Consistently, application of the A1R agonist N6-cyclohexyladenosine (1 µM) recapitulated the inhibitory action of adenosine on ClC-K2/b open probability. The effects of adenosine signaling in the collecting duct were independent from its purinergic counterpartner, ATP, having no measurable actions on ClC-K2/b and Kir4.1/5.1. Overall, we demonstrated that adenosine selectively inhibits ClC-K2/b activity in intercalated cells by targeting A1Rs. We propose that inhibition of transcellular Cl- reabsorption in the collecting duct by adenosine would aid in augmenting NaCl excretion during high salt intake.

TRPV4 deletion protects against hypokalemia during systemic K+ deficiency.

Tight regulation of K+ balance is fundamental for normal physiology. Reduced dietary K+ intake, which is common in Western diets, often leads to hypokalemia and associated cardiovascular- and kidney-related pathologies. The distal nephron, and, specifically, the collecting duct (CD), is the major site of controlled K+ reabsorption via H+-K+-ATPase in the state of dietary K+ deficiency. We (Mamenko MV, Boukelmoune N, Tomilin VN, Zaika OL, Jensen VB, O'Neil RG, Pochynyuk OM. Kidney Int 91: 1398-1409, 2017) have previously demonstrated that the transient receptor potential vanilloid type 4 (TRPV4) Ca2+ channel, abundantly expressed in the CD, contributes to renal K+ handling by promoting flow-induced K+ secretion. Here, we investigated a potential role of TRPV4 in controlling H+-K+-ATPase-dependent K+ reabsorption in the CD. Treatment with a K+-deficient diet (<0.01% K+) for 7 days reduced serum K+ levels in wild-type (WT) mice from 4.3 ± 0.2 to 3.3 ± 0.2 mM but not in TRPV4-/- mice (4.3 ± 0.1 and 4.2 ± 0.3 mM, respectively). Furthermore, we detected a significant reduction in 24-h urinary K+ levels in TRPV4-/- compared with WT mice upon switching to K+-deficient diet. TRPV4-/- animals also had significantly more acidic urine on a low-K+ diet, but not on a regular (0.9% K+) or high-K+ (5% K+) diet, which is consistent with increased H+-K+-ATPase activity. Moreover, we detected a greatly accelerated H+-K+-ATPase-dependent intracellular pH extrusion in freshly isolated CDs from TRPV4-/- compared with WT mice fed a K+-deficient diet. Overall, our results demonstrate a novel kaliuretic role of TRPV4 by inhibiting H+-K+-ATPase-dependent K+ reabsorption in the CD. We propose that TRPV4 inhibition could be a novel strategy to manage certain hypokalemic states in clinical settings.

Deficient transient receptor potential vanilloid type 4 function contributes to compromised [Ca2+]i homeostasis in human autosomal-dominant polycystic kidney disease cells.

Autosomal-dominant polycystic kidney disease (ADPKD) is a devastating disorder that is characterized by a progressive decline in renal function as a result of the development of fluid-filled cysts. Defective flow-mediated [Ca2+]i responses and disrupted [Ca2+]i homeostasis have been repeatedly associated with cyst progression in ADPKD. We have previously demonstrated that the transient receptor potential vanilloid type 4 (TRPV4) channel is imperative for flow-mediated [Ca2+]i responses in murine distal renal tubule cells. To determine whether compromised TRPV4 function contributes to aberrant Ca2+ regulation in ADPKD, we assessed TRPV4 function in primary cells that were cultured from ADPKD and normal human kidneys (NHKs). Single-channel TRPV4 activity and TRPV4-dependent Ca2+ influxes were drastically reduced in ADPKD cells, which correlated with distorted [Ca2+]i signaling. Whereas total TRPV4 protein levels were comparable in NHK and ADPKD cells, we detected a marked decrease in TRPV4 glycosylation in ADPKD cells. Tunicamycin-induced deglycosylation inhibited TRPV4 activity and compromised [Ca2+]i signaling in NHK cells. Overall, we demonstrate that TRPV4 glycosylation and channel activity are diminished in human ADPKD cells compared with NHK cells, and that this contributes significantly to the distorted [Ca2+]i dynamics. We propose that TRPV4 stimulation may be beneficial for restoring [Ca2+]i homeostasis in cyst cells, thereby interfering with ADPKD progression.-Tomilin, V., Reif, G. A., Zaika, O., Wallace, D. P., Pochynyuk, O. Deficient transient receptor potential vanilloid type 4 function contributes to compromised [Ca2+]i homeostasis in human autosomal-dominant polycystic kidney disease cells.

Sustained Elevated Adenosine via ADORA2B Promotes Chronic Pain through Neuro-immune Interaction.

The molecular mechanisms of chronic pain are poorly understood and effective mechanism-based treatments are lacking. Here, we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected chronic mechanical and thermal hypersensitivity due to sustained elevated circulating adenosine. Extending from Ada(-/-) mice, we further discovered that prolonged elevated adenosine contributed to chronic pain behaviors in two additional independent animal models: sickle cell disease mice, a model of severe pain with limited treatment, and complete Freund's adjuvant paw-injected mice, a well-accepted inflammatory model of chronic pain. Mechanistically, we revealed that activation of adenosine A2B receptors on myeloid cells caused nociceptor hyperexcitability and promoted chronic pain via soluble IL-6 receptor trans-signaling, and our findings determined that prolonged accumulated circulating adenosine contributes to chronic pain by promoting immune-neuronal interaction and revealed multiple therapeutic targets.

Discrete control of TRPV4 channel function in the distal nephron by protein kinases A and C.

We have recently documented that the Ca(2+)-permeable TRPV4 channel, which is abundantly expressed in distal nephron cells, mediates cellular Ca(2+) responses to elevated luminal flow. In this study, we combined Fura-2-based [Ca(2+)]i imaging with immunofluorescence microscopy in isolated split-opened distal nephrons of C57BL/6 mice to probe the molecular determinants of TRPV4 activity and subcellular distribution. We found that activation of the PKC pathway with phorbol 12-myristate 13-acetate significantly increased [Ca(2+)]i responses to flow without affecting the subcellular distribution of TRPV4. Inhibition of PKC with bisindolylmaleimide I diminished cellular responses to elevated flow. In contrast, activation of the PKA pathway with forskolin did not affect TRPV4-mediated [Ca(2+)]i responses to flow but markedly shifted the subcellular distribution of the channel toward the apical membrane. These actions were blocked with the specific PKA inhibitor H-89. Concomitant activation of the PKA and PKC cascades additively enhanced the amplitude of flow-induced [Ca(2+)]i responses and greatly increased basal [Ca(2+)]i levels, indicating constitutive TRPV4 activation. This effect was precluded by the selective TRPV4 antagonist HC-067047. Therefore, the functional status of the TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. Although the PKA-dependent cascade promotes TRPV4 trafficking and translocation to the apical membrane, the PKC-dependent pathway increases the activity of the channel on the plasma membrane.

Primary aldosteronism and impaired natriuresis in mice underexpressing TGFß1.

To uncover the potential cardiovascular effects of human polymorphisms influencing transforming growth factor ß1 (TGFß1) expression, we generated mice with Tgfb1 mRNA expression graded in five steps from 10% to 300% normal. Adrenal expression of the genes for mineralocorticoid-producing enzymes ranged from 50% normal in the hypermorphs at age 12 wk to 400% normal in the hypomorphs accompanied with proportionate changes in plasma aldosterone levels, whereas plasma volumes ranged from 50% to 150% normal accompanied by marked compensatory changes in plasma angiotensin II and renin levels. The aldosterone/renin ratio ranged from 0.3 times normal in the 300% hypermorphs to six times in the 10% hypomorphs, which have elevated blood pressure. Urinary output of water and electrolytes are markedly decreased in the 10% hypomorphs without significant change in the glomerular filtration rate. Renal activities for the Na(+), K(+)-ATPase, and epithelial sodium channel are markedly increased in the 10% hypomorphs. The hypertension in the 10% hypomorphs is corrected by spironolactone or amiloride at doses that do not change blood pressure in wild-type mice. Thus, changes in Tgfb1 expression cause marked progressive changes in multiple systems that regulate blood pressure and fluid homeostasis, with the major effects being mediated by changes in adrenocortical function.

TRPV4 dysfunction promotes renal cystogenesis in autosomal recessive polycystic kidney disease.

The molecular mechanism of cyst formation and expansion in autosomal recessive polycystic kidney disease (ARPKD) is poorly understood, but impaired mechanosensitivity to tubular flow and dysfunctional calcium signaling are important contributors. The activity of the mechanosensitive Ca(2+)-permeable TRPV4 channel underlies flow-dependent Ca(2+) signaling in murine collecting duct (CD) cells, suggesting that this channel may contribute to cystogenesis in ARPKD. Here, we developed a method to isolate CD-derived cysts and studied TRPV4 function in these cysts laid open as monolayers and in nondilated split-open CDs in a rat model of ARPKD. In freshly isolated CD-derived cyst monolayers, we observed markedly impaired TRPV4 activity, abnormal subcellular localization of the channel, disrupted TRPV4 glycosylation, decreased basal [Ca(2+)]i, and loss of flow-mediated [Ca(2+)]i signaling. In contrast, nondilated CDs of these rats exhibited functional TRPV4 with largely preserved mechanosensitive properties. Long-term systemic augmentation of TRPV4 activity with a selective TRPV4 activator significantly attenuated the renal manifestations of ARPKD in a time-dependent manner. At the cellular level, selective activation of TRPV4 restored mechanosensitive Ca(2+) signaling as well as the function and subcellular distribution of TRPV4. In conclusion, the functional status of TRPV4, which underlies mechanosensitive Ca(2+) signaling in CD cells, inversely correlates with renal cystogenesis in ARPKD. Augmenting TRPV4 activity may have therapeutic potential in ARPKD.

Purinergic activation of Ca2+-permeable TRPV4 channels is essential for mechano-sensitivity in the aldosterone-sensitive distal nephron.

Mechanical forces are known to induce increases of [Ca(2+)](i) in the aldosterone-sensitive distal nephron (ASDN) cells to regulate epithelial transport. At the same time, mechanical stress stimulates ATP release from ASDN cells. In this study, we combined ratiometric Fura-2 based monitoring of [Ca(2+)](i) in freshly isolated split-opened ASDN with targeted deletion of P2Y2 and TRPV4 in mice to probe a role for purinergic signaling in mediating mechano-sensitive responses in ASDN cells. ATP application causes a reproducible transient Ca(2+) peak followed by a sustained plateau. Individual cells of the cortical collecting duct (CCD) and the connecting tubule (CNT) respond to purinergic stimulation with comparative elevations of [Ca(2+)](i). Furthermore, ATP-induced Ca(2+)-responses are nearly identical in both principal (AQP2-positive) and intercalated (AQP2-negative) cells as was confirmed using immunohistochemistry in split-opened ASDN. UTP application produces elevations of [Ca(2+)](i) similar to that observed with ATP suggesting a dominant role of P2Y2-like receptors in generation of [Ca(2+)](i) response. Indeed, genetic deletion of P2Y2 receptors decreases the magnitude of ATP-induced and UTP-induced Ca(2+) responses by more than 70% and 90%, respectively. Both intracellular and extracellular sources of Ca(2+) appeared to contribute to the generation of ATP-induced Ca(2+) response in ASDN cells. Importantly, flow- and hypotonic-induced Ca(2+) elevations are markedly blunted in P2Y2 -/- mice. We further demonstrated that activation of mechano-sensitive TRPV4 channel plays a major role in the sustained [Ca(2+)](i) elevation during purinergic stimulation. Consistent with this, ATP-induced Ca(2+) plateau are dramatically attenuated in TRV4 -/- mice. Inhibition of TRPC channels with 10 µM BTP2 also decreased ATP-induced Ca(2+) plateau whilst to a lower degree than that observed with TRPV4 inhibition/genetic deletion. We conclude that stimulation of purinergic signaling by mechanical stimuli leads to activation of TRPV4 and, to a lesser extent, TRPCs channels, and this is an important component of mechano-sensitive response of the ASDN.

Af17 deficiency increases sodium excretion and decreases blood pressure.

The putative transcription factor AF17 upregulates the transcription of the epithelial sodium channel (ENaC) genes, but whether AF17 modulates sodium homeostasis and BP is unknown. Here, we generated Af17-deficient mice to determine whether deletion of Af17 leads to sodium wasting and low BP. Compared with wild-type mice, Af17-deficient mice had lower BP (11 mmHg), higher urine volume, and increased sodium excretion despite mildly increased plasma concentrations of aldosterone. Deletion of Af17 led to increased dimethylation of histone H3 K79 and reduced ENaC function. The attenuated function of ENaC resulted from decreased ENaC mRNA and protein expression, fewer active channels, lower open probability, and reduced effective activity. In contrast, inducing high levels of plasma aldosterone by a variety of methods completely compensated for Af17 deficiency with respect to sodium handling and BP. Taken together, these data identify Af17 as a potential locus for the maintenance of sodium and BP homeostasis and suggest that a particular histone modification is directly linked to these processes. Af17-mediated regulation of BP is largely, but not exclusively, the result of modulating ENaC, suggesting it has potential as a therapeutic target for the control of BP.

Functional role of M-type (KCNQ) K⁺ channels in adrenergic control of cardiomyocyte contraction rate by sympathetic neurons.

M-type (KCNQ) K⁺ channels are known to regulate excitability and firing properties of sympathetic neurons (SNs), but their role in regulating neurotransmitter release is unclear, requiring further study. We sought to use a physiological preparation in which SNs innervate primary cardiomyocytes to evaluate the direct role of M-channels in the release of noradrenaline (NA) from SNs. Co-cultures of rat SNs and mouse cardiomyocytes were prepared, and the contraction rate (CR) of the cardiomyocyte syncytium monitored by video microscopy. We excited the SNs with nicotine, acting on nicotinic acetylcholine receptors, and monitored the increase in CR in the presence or absence of the specific M-channel opener retigabine, or agonists of bradykinin B2 or purinergic P2Y receptors on the SNs. The maximal adrenergic effect on the CR was determined by application of isoproterenol (isoprenaline). To isolate the actions of B2 or P2Y receptor stimulation to the neurons, we prepared cardiomyocytes from B2 receptor or P2Y2 receptor knock-out mice, respectively. We found that co-application of retigabine strongly decreased the nicotine-induced increase in CR. Conversely, co-application of bradykinin or the P2Y-receptor agonist UTP augmented the nicotine-induced increase in CR to about half of the level produced by isoproterenol. All effects on the CR were wholly blocked by propranolol. Our data support the role of M-type K⁺ channels in the control of NA release by SNs at functional adrenergic synapses on cardiomyocytes.We conclude that physiological receptor agonists control the heart rate via the regulation of M-current in SNs.

Bradykinin acutely inhibits activity of the epithelial Na+ channel in mammalian aldosterone-sensitive distal nephron.

Activation of the renal kallikrein-kinin system results in natriuresis and diuresis, suggesting its possible role in renal tubular sodium transport regulation. Here, we used patch-clamp electrophysiology to directly assess the effects of bradykinin (BK) on the epithelial Na(+) channel (ENaC) activity in freshly isolated split-opened murine aldosterone-sensitive distal nephrons (ASDNs). BK acutely inhibits ENaC activity by reducing channel open probability (P(o)) in a dose-dependent and reversible manner. Inhibition of B2 receptors with icatibant (HOE-140) abolished BK actions on ENaC. In contrast, activation of B1 receptors with the selective agonist Lys-des-Arg(9)-BK failed to reproduce BK actions on ENaC. This is consistent with B2 receptors playing a critical role in mediating BK signaling to ENaC. BK has little effect on ENaC P(o) when G(q/11) was inhibited with Gp antagonist 2A. Moreover, inhibition of phospholipase C (PLC) with U73122, but not saturation of cellular cAMP levels with the membrane-permeable nonhydrolysable cAMP analog 8-cpt-cAMP, prevents BK actions on ENaC activity. This argues that BK stimulates B2 receptors with subsequent activation of G(q/11)-PLC signaling cascade to acutely inhibit ENaC activity. Activation of BK signaling acutely depletes apical PI(4,5)P(2) levels. However, inhibition of Ca(2+) pump SERCA of the endoplasmic reticulum with thapsigargin does not prevent BK signaling to ENaC. Furthermore, caffeine, while producing a similar rise in [Ca(2+)](i) as in response to BK stimulation, fails to recapitulate BK actions on ENaC. Therefore, we concluded that BK acutely inhibits ENaC P(o) in mammalian ASDN via stimulation of B2 receptors and following depletion of PI(4,5)P(2), but not increases in [Ca(2+)](i).

Oxidative modification of M-type K(+) channels as a mechanism of cytoprotective neuronal silencing.

Voltage-gated K(+) channels of the Kv7 family underlie the neuronal M current that regulates action potential firing. Suppression of M current increases excitability and its enhancement can silence neurons. We here show that three of five Kv7 channels undergo strong enhancement of their activity by oxidative modification induced by physiological concentrations of hydrogen peroxide. A triple cysteine pocket in the channel S2-S3 linker is critical for this effect. Oxidation-induced enhancement of M current produced a hyperpolarization and a dramatic reduction of action potential firing frequency in rat sympathetic neurons. As hydrogen peroxide is robustly produced during hypoxia-induced oxidative stress, we used an oxygen/glucose deprivation neurodegeneration model that showed neuronal death to be severely accelerated by M current blockade. Such blockade had no effect on survival of normoxic neurons. This work describes a novel pathway of M-channel regulation and suggests a role for M channels in protective neuronal silencing during oxidative stress.