TRPC3 determines osmosensitive [Ca2+]i signaling in the collecting duct and contributes to urinary concentration

It is well-established that the kidney collecting duct (CD) plays a central role in regulation of systemic water homeostasis. Aquaporin 2 (AQP2)-dependent water reabsorption in the CD critically depends on the arginine vasopressin (AVP) antidiuretic input and the presence of a favorable osmotic gradient at the apical plasma membrane with tubular lumen being hypotonic compared to the cytosol. This osmotic difference creates a mechanical force leading to an increase in [Ca2+]i in CD cells. The significance of the osmosensitive [Ca2+]i signaling for renal water transport and urinary concentration remain unknown. To examine molecular mechanism and physiological relevance of osmosensitivity in the CD, we implemented simultaneous direct measurements of [Ca2+]i dynamics and the rate of cell swelling as a readout of the AQP2-dependent water reabsorption in freshly isolated split-opened CDs of wild type and genetically manipulated animals and combined this with immunofluorescent detection of AVP-induced AQP2 trafficking and assessment of systemic water balance. We identified the critical role of the Ca2+-permeable TRPC3 channel in osmosensitivity and water permeability in the CD. We further demonstrated that TRPC3 -/- mice exhibit impaired urinary concentration, larger urinary volume and a greater weight loss in response to water deprivation despite increased AVP levels and AQP2 abundance. TRPC3 deletion interfered with AQP2 translocation to the plasma membrane in response to water deprivation. In summary, we provide compelling multicomponent evidence in support of a critical contribution of TRPC3 in the CD for osmosensitivity and renal water handling.Fil: Tomilin, Viktor N.. University of Texas; Estados UnidosFil: Mamenko, Mykola. Augusta University; Estados UnidosFil: Zaika, Oleg. University of Texas; Estados UnidosFil: Ren, Guohui. University of Texas; Estados UnidosFil: Marrelli, Sean P.. University of Texas; Estados UnidosFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Pochynyuk, Oleh. University of Texas; Estados Unido

Emerging role of the calcium-activated, small conductance, SK3 K ⁺ channel in distal tubule function: Regulation by TRPV4

The Ca2+-activated, maxi-K (BK) K+ channel, with low Ca2+-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K+ secretion. In the present study we demonstrate that the Ca2+-activated, SK3 (KCa2.3) K + channel, with high Ca2+-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K+ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca2+- dependent K+ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca2+-permeable TRPV4 channel, thereby inducing Ca2+ influx and elevating intracellular Ca2+ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+- affinity K+ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca2+-dependent regulation of membrane potential and K+ secretion. © 2014 Berrout et al

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

Ca²⁺-imaging in aldosterone-sensitive distal nephron.

<p>Representative micrographs of split-opened cortical collecting duct (top raw) and connecting tubules (bottom raw) after loading with Fura-2 taken with bright-field illumination (left column), 380 nm excitation (middle column), and the merged image (right column).</p

ATP increases [Ca²⁺]_i in a PLC-dependent manner.

<p>(<b>A</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min ATP applications (shown with gray bar on the top) for individual cells of ASDN in the absence and presence of a PLC inhibitor, U73122 (black bar). (<b>B</b>) Summary graph of the ATP-induced changes in ΔF₃₄₀/F₃₈₀ in the control and after PLC inhibition. * - significant decrease versus ATP. (<b>C</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min ATP applications (shown with gray bar on the top) for individual cells of ASDN in the absence and presence of a PLA inhibitor, AACOCF3 (black bar). (<b>D</b>) Summary graph of the ATP-induced changes in ΔF₃₄₀/F₃₈₀ in the control and after PLA inhibition. * - significant decrease versus ATP.</p

Extracellular and intracellular Ca²⁺ sources account for ATP-evoked [Ca²⁺]_i elevations.

<p>(<b>A</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min ATP applications (shown with a gray bar on the top) for individual cells of ASDN in the absence and presence of a Ca²⁺-pump SERCA inhibitor, thapsigargin (black bar). (<b>B</b>) Summary graph of the ATP-induced changes in ΔF₃₄₀/F₃₈₀ in the control and after SERCA inhibition. * - significant decrease versus ATP. (<b>C</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min ATP applications (shown with gray bar on the top) for individual cells of ASDN in the control and in Ca²⁺-free extracellular media (black bar). (<b>D</b>) Summary graph of the ATP-induced changes in ΔF₃₄₀/F₃₈₀ in the control and after extracellular Ca²⁺ removal. * - significant decrease versus ATP.</p

Disruption of purinergic signaling compromises mechano-sensitive responses in ASDN cells.

<p>(<b>A</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min of hypotonic (hypo) media application (shown with bar on the top) for individual cells of ASDN isolated from wild type (wt, black) and P2Y2 −/− (light gray) mice. (<b>B</b>) Summary graph of the ΔF₃₄₀/F₃₈₀ peak changes in response to hypotonic media application for wild type and P2Y2 −/− mice. * - significant decrease versus hypo WT. (<b>C</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to elevated flow (shown with bar on the top) for individual cells of ASDN from wild type (black) and P2Y2 −/− (light gray) mice. (<b>D</b>) Summary graph of the magnitudes of high flow-induced Ca²⁺ spikes for wild type and P2Y2 −/− mice. * - significant decrease versus flow WT.</p

TRPV4 is critical for the ATP-induced Ca²⁺-plateau.

<p>(<b>A</b>) The average time course of relative changes in ΔF₃₄₀/F₃₈₀ in response to 2 min ATP application (shown with bar on the top) for individual cells of ASDN from wild type (black) and TRPV4 −/− (gray) mice. (<b>B</b>) Summary graph of relative contributions of TRPV4 and TRPCs in ATP-induced [Ca²⁺]_i response. For this, the values of ΔF₃₄₀/F₃₈₀ during second ATP application from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022824#pone-0022824-g007" target="_blank">Figures 7A, B</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022824#pone-0022824-g008" target="_blank">8A</a> were subtracted from the corresponding values during the first ATP application.</p

Urinary concentrating defect in mice lacking Epac1 or Epac2

cAMP is a universal second messenger regulating a plethora of processes in the kidney. Two downstream effectors of cAMP are PKA and exchange protein directly activated by cAMP (Epac), which, unlike PKA, is often linked to elevation of [Ca2+]i. While both Epac isoforms (Epac1 and Epac2) are expressed along the nephron, their relevance in the kidney remains obscure. We combined ratiometric calcium imaging with quantitative immunoblotting, immunofluorescent confocal microscopy, and balance studies in mice lacking Epac1 or Epac2 to determine the role of Epac in renal water-solute handling. Epac1-/- and Epac2-/- mice developed polyuria despite elevated arginine vasopressin levels. We did not detect major deficiencies in arginine vasopressin [Ca2+]i signaling in split-opened collecting ducts or decreases in aquaporin water channel type 2 levels. Instead, sodium-hydrogen exchanger type 3 levels in the proximal tubule were dramatically reduced in Epac1-/- and Epac2-/- mice. Water deprivation revealed persisting polyuria, impaired urinary concentration ability, and augmented urinary excretion of Na+ and urea in both mutant mice. In summary, we report a nonredundant contribution of Epac isoforms to renal function. Deletion of Epac1 and Epac2 decreases sodium-hydrogen exchanger type 3 expression in the proximal tubule, leading to polyuria and osmotic diuresis.-Cherezova, A., Tomilin, V., Buncha, V., Zaika, O., Ortiz, P. A., Mei, F., Cheng, X., Mamenko, M., Pochynyuk, O. Urinary concentrating defect in mice lacking Epac1 or Epac2