Mykola Mamenko

Angiotensin II increases activity of the epithelial Na+ channel (ENaC) in distal nephron additively to aldosterone.

Background: Mineralocorticoid aldosterone controls ENaC-mediated Na+ reabsorption in the distal nephron. Results: Ang II stimulates production of reactive oxygen species to stimulate ENaC, and this effect is preserved when mineralocorticoids are high. Conclusion: Ang II directly controls ENaC activity and expression in murine distal nephrons. Significance: Ang II may have a specific role in regulation of sodium handling in the distal nephron during variations in dietary Na+ intake. Dietary salt intake controls epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in the distal nephron by affecting status of the renin-angiotensin-aldosterone system (RAAS). Whereas regulation of ENaC by aldosterone is generally accepted, little is known about whether other components of RAAS, such as angiotensin II (Ang II), have nonredundant to aldosterone-stimulatory actions on ENaC. We combined patch clamp electrophysiology and immunohistochemistry in freshly isolated split-opened distal nephrons of mice to determine the mechanism and molecular signaling pathway of Ang II regulation of ENaC. We found that Ang II acutely increases ENaC Po, whereas prolonged exposure to Ang II also induces translocation of α-ENaC toward the apical membrane in situ. Ang II actions on ENaC Po persist in the presence of saturated mineralocorticoid status. Moreover, aldosterone fails to stimulate ENaC acutely, suggesting that Ang II and aldosterone have different time frames of ENaC activation. AT1 but not AT2 receptors mediate Ang II actions on ENaC. Unlike its effect in vasculature, Ang II did not increase [Ca2+]i in split-opened distal nephrons as demonstrated using ratiometric Fura-2-based microscopy. However, application of Ang II to mpkCCDc14 cells resulted in generation of reactive oxygen species, as probed with fluorescent methods. Consistently, inhibiting NADPH oxidase with apocynin abolished Ang II-mediated increases in ENaC Po in murine distal nephron. Therefore, we concluded that Ang II directly regulates ENaC activity in the distal nephron, and this effect complements regulation of ENaC by aldosterone. We propose that stimulation of AT1 receptors with subsequent activation of NADPH oxidase signaling pathway mediates Ang II actions on ENaC.

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).

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 [Ca2+]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 [Ca2+]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 Ca2+ 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 [Ca2+]i. Furthermore, ATP-induced Ca2+-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 [Ca2+]i similar to that observed with ATP suggesting a dominant role of P2Y2-like receptors in generation of [Ca2+]i response. Indeed, genetic deletion of P2Y2 receptors decreases the magnitude of ATP-induced and UTP-induced Ca2+ responses by more than 70% and 90%, respectively. Both intracellular and extracellular sources of Ca2+ appeared to contribute to the generation of ATP-induced Ca2+ response in ASDN cells. Importantly, flow- and hypotonic-induced Ca2+ elevations are markedly blunted in P2Y2 −/− mice. We further demonstrated that activation of mechano-sensitive TRPV4 channel plays a major role in the sustained [Ca2+]i elevation during purinergic stimulation. Consistent with this, ATP-induced Ca2+ plateau are dramatically attenuated in TRV4 −/− mice. Inhibition of TRPC channels with 10 µM BTP2 also decreased ATP-induced Ca2+ 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.

Chronic angiotensin II infusion drives extensive aldosterone-independent epithelial Na+ channel activation

The inability of mineralocorticoid receptor (MR) blockade to reduce hypertension associated with high angiotensin (Ang) II suggests direct actions of Ang II to regulate tubular sodium reabsorption via the epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron. We used freshly isolated aldosterone-sensitive distal nephron from mice to delineate the synergism and primacy between aldosterone and Ang II in controlling functional ENaC activity. Inhibition of MR specifically prevented the increased number of functionally active ENaC, but not ENaC open probability elicited by a low sodium diet. In contrast, we found no functional role of glucocorticoid receptors in the regulation of ENaC activity by dietary salt intake. Simultaneous inhibition of MR and Ang II type 1 receptors ameliorated the enhanced ENaC activity caused by low dietary salt intake and produced significantly greater natriuresis than either inhibitor alone. Chronic systemic Ang II infusion induced more than 2 times greater increase in ENaC activity than observed during dietary sodium restriction. Importantly, ENaC activity remained greatly above control levels during maximal MR inhibition. We conclude that during variations in dietary salt intake both aldosterone and Ang II contribute complementarily to the regulation of ENaC activity in the aldosterone-sensitive distal nephron. In contrast, in the setting of Ang II–dependent hypertension, ENaC activity is upregulated well above the physiological range and is not effectively suppressed by inhibition of the aldosterone–MR axis. This provides a mechanistic explanation for the resistance to MR inhibition that occurs in hypertensive subjects having elevated intrarenal Ang II levels.

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.

Chronic Angiotensin II Infusion Drives Extensive Aldosterone-Independent Epithelial Na+ Channel ActivationNovelty and Significance

The inability of mineralocorticoid receptor (MR) blockade to reduce hypertension associated with high angiotensin (Ang) II suggests direct actions of Ang II to regulate tubular sodium reabsorption via the epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron. We used freshly isolated aldosterone-sensitive distal nephron from mice to delineate the synergism and primacy between aldosterone and Ang II in controlling functional ENaC activity. Inhibition of MR specifically prevented the increased number of functionally active ENaC, but not ENaC open probability elicited by a low sodium diet. In contrast, we found no functional role of glucocorticoid receptors in the regulation of ENaC activity by dietary salt intake. Simultaneous inhibition of MR and Ang II type 1 receptors ameliorated the enhanced ENaC activity caused by low dietary salt intake and produced significantly greater natriuresis than either inhibitor alone. Chronic systemic Ang II infusion induced more than 2 times greater increase in ENaC activity than observed during dietary sodium restriction. Importantly, ENaC activity remained greatly above control levels during maximal MR inhibition. We conclude that during variations in dietary salt intake both aldosterone and Ang II contribute complementarily to the regulation of ENaC activity in the aldosterone-sensitive distal nephron. In contrast, in the setting of Ang II–dependent hypertension, ENaC activity is upregulated well above the physiological range and is not effectively suppressed by inhibition of the aldosterone–MR axis. This provides a mechanistic explanation for the resistance to MR inhibition that occurs in hypertensive subjects having elevated intrarenal Ang II levels.

Salt-dependent inhibition of epithelial Na+ channel-mediated sodium reabsorption in the aldosterone-sensitive distal nephron by bradykinin

We have documented recently that bradykinin (BK) directly inhibits activity of the epithelial Na+ channel (ENaC) via the bradykinin B2 receptor (B2R)-Gq/11-phospholipase C pathway. In this study, we took advantage of mice genetically engineered to lack bradykinin receptors (B1R, B2R−/−) to probe a physiological role of BK cascade in regulation of ENaC in native tissue, aldosterone-sensitive distal nephron. Under normal sodium intake (0.32% Na+), ENaC open probability (Po) was modestly elevated in B1R, B2R−/− mice compared with wild-type mice. This difference is augmented during elevated Na+ intake (2.00% Na+) and negated during Na+ restriction (<0.01% Na+). Saturation of systemic mineralocorticoid status with deoxycorticosterone acetate similarly increased ENaC activity in both mouse strains, suggesting that the effect of BK on ENaC is independent of aldosterone. It is accepted that angiotensin-converting enzyme represents the major pathway of BK degradation. Systemic inhibition of angiotensin-converting enzyme with captopril (30 mg/kg of body weight for 7 days) significantly decreases ENaC activity and Po in wild-type mice, but this effect is diminished in B1R, B2R−/− mice. At the cellular level, acute captopril (100 &mgr;mol/L) treatment sensitized BK signaling cascade and greatly potentiated the inhibitory effect of 100 nmol/L of BK on ENaC. We concluded that BK cascade has its own specific role in blunting ENaC activity, particularly under conditions of elevated sodium intake. Augmentation of BK signaling in the aldosterone-sensitive distal nephron inhibits ENaC-mediated Na+ reabsorption, contributing to the natriuretic and antihypertensive effects of angiotensin-converting enzyme inhibition.

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

Background: TRPV4 mediates flow-induced [Ca2+]i responses in distal nephron cells. Results: Activation of PKC augments TRPV4-mediated responses to flow. Activation of PKA promotes TRPV4 translocation to the apical membrane. Conclusion: TRPV4 activity and TRPV4 trafficking are under discrete but synergistic control of PKC- and PKA-dependent pathways. Significance: Systemic physiological stimuli may affect TRPV4-mediated mechanosensitivity in the distal nephron via PKA- and PKC-dependent mechanisms. We have recently documented that the Ca2+-permeable TRPV4 channel, which is abundantly expressed in distal nephron cells, mediates cellular Ca2+ responses to elevated luminal flow. In this study, we combined Fura-2-based [Ca2+]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 [Ca2+]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 [Ca2+]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 [Ca2+]i responses and greatly increased basal [Ca2+]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.

Direct Activation of ENaC by Angiotensin II: Recent Advances and New Insights

Angiotensin II (Ang II) is the principal effector of the renin–angiotensin–aldosterone system (RAAS). It initiates myriad processes in multiple organs integrated to increase circulating volume and elevate systemic blood pressure. In the kidney, Ang II stimulates renal tubular water and salt reabsorption causing antinatriuresis and antidiuresis. Activation of the RAAS is known to enhance activity of the epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron. In addition to its well described stimulatory actions on aldosterone secretion, Ang II is also capable of directly increasing ENaC activity. In this brief review, we discuss recent findings about non-classical Ang II actions on ENaC and speculate about its relevance for renal sodium handling.

Direct inhibition of basolateral Kir4.1/5.1 and Kir4.1 channels in the cortical collecting duct by dopamine

It is recognized that dopamine promotes natriuresis by inhibiting multiple transporting systems in the proximal tubule. In contrast, less is known about the molecular targets of dopamine actions on water-electrolyte transport in the cortical collecting duct (CCD). Epithelial cells in the CCD are exposed to dopamine, which is synthesized locally or secreted from sympathetic nerve endings. Basolateral K(+) channels in the distal renal tubule are critical for K(+) recycling and controlling basolateral membrane potential to establish the driving force for Na(+) reabsorption. Here, we demonstrate that Kir4.1 and Kir5.1 are highly expressed in the mouse kidney cortex and are localized to the basolateral membrane of the CCD. Using patch-clamp electrophysiology in freshly isolated CCDs, we detected highly abundant 40-pS and scarce 20-pS single channel conductances, most likely representing Kir4.1/5.1 and Kir4.1 channels, respectively. Dopamine reversibly decreased the open probability of both channels, with a relatively greater action on the Kir4.1/5.1 heterodimer. This effect was mediated by D2-like but not D1-like dopamine receptors. PKC blockade abolished the inhibition of basolateral K(+) channels by dopamine. Importantly, dopamine significantly decreased the amplitude of Kir4.1/5.1 and Kir4.1 unitary currents. Consistently, dopamine induced an acute depolarization of basolateral membrane potential, as directly monitored using current-clamp mode in isolated CCDs. Therefore, we demonstrate that dopamine inhibits basolateral Kir4.1/5.1 and Kir4.1 channels in CCD cells via stimulation of D2-like receptors and subsequently PKC. This leads to depolarization of the basolateral membrane and a decreased driving force for Na(+) reabsorption in the distal renal tubule.