Cortical Collecting Duct Principal Cells Research Articles

MicroRNA-19 is regulated by aldosterone in a sex-specific manner to alter kidney sodium transport.

A key regulator of blood pressure homeostasis is the steroid hormone aldosterone, which is released as the final signaling hormone of the renin-angiotensin-aldosterone-signaling (RAAS) system. Aldosterone increases sodium (Na+) reabsorption in the kidney distal nephron to regulate blood volume. Unregulated RAAS signaling can lead to hypertension and cardiovascular disease. The serum and glucocorticoid kinase (SGK1) coordinates much of the Na+ reabsorption in the cortical collecting duct (CCD) tubular epithelial cells. We previously demonstrated that aldosterone alters the expression of microRNAs (miRs) in CCD principal cells. The aldosterone-regulated miRs can modulate Na+ transport and the cellular response to aldosterone signaling. However, the sex-specific regulation of miRs by aldosterone in the kidney distal nephron has not been explored. In this study, we report that miR-19, part of the miR-17-92 cluster, is upregulated in female mouse CCD cells in response to aldosterone activation. Mir-19 binding to the 3'-untranslated region of SGK1 was confirmed using a dual-luciferase reporter assay. Increasing miR-19 expression in CCD cells decreased SGK1 message and protein expression. Removal of this cluster using a nephron-specific, inducible knockout mouse model increased SGK1 expression in female mouse CCD cells. The miR-19-induced decrease in SGK1 protein expression reduced the response to aldosterone stimulation and may account for sex-specific differences in aldosterone signaling. By examining evolution of the miR-17-92 cluster, phylogenetic sequence analysis indicated that this cluster arose at the same time that other Na+-sparing and salt regulatory proteins, specifically SGK1, first emerged, indicating a conserved role for these miRs in kidney function of salt and water homeostasis.NEW & NOTEWORTHY Expression of the microRNA-17-92 cluster is upregulated by aldosterone in mouse cortical collecting duct principal cells, exclusively in female mice. MiR-19 in this cluster targets the serum and glucocorticoid kinase (SGK1) to downregulate both mRNA and protein expression, resulting in a decrease in sodium transport across epithelial cells of the collecting duct. The miR-17-92 cluster is evolutionarily conserved and may act as a novel feedback regulator for aldosterone signaling in females.

Prostaglandin E2 stimulates the epithelial sodium channel (ENaC) in cultured mouse cortical collecting duct cells in an autocrine manner.

Prostaglandin E2 (PGE2) is the most abundant prostanoid in the kidney, affecting a wide range of renal functions. Conflicting data have been reported regarding the effects of PGE2 on tubular water and ion transport. The amiloride-sensitive epithelial sodium channel (ENaC) is rate limiting for transepithelial sodium transport in the aldosterone-sensitive distal nephron. The aim of the present study was to explore a potential role of PGE2 in regulating ENaC in cortical collecting duct (CCD) cells. Short-circuit current (ISC) measurements were performed using the murine mCCDcl1 cell line known to express characteristic properties of CCD principal cells and to be responsive to physiological concentrations of aldosterone and vasopressin. PGE2 stimulated amiloride-sensitive ISC via basolateral prostaglandin E receptors type 4 (EP4) with an EC50 of ∼7.1 nM. The rapid stimulatory effect of PGE2 on ISC resembled that of vasopressin. A maximum response was reached within minutes, coinciding with an increased abundance of β-ENaC at the apical plasma membrane and elevated cytosolic cAMP levels. The effects of PGE2 and vasopressin were nonadditive, indicating similar signaling cascades. Exposing mCCDcl1 cells to aldosterone caused a much slower (∼2 h) increase of the amiloride-sensitive ISC. Interestingly, the rapid effect of PGE2 was preserved even after aldosterone stimulation. Furthermore, application of arachidonic acid also increased the amiloride-sensitive ISC involving basolateral EP4 receptors. Exposure to arachidonic acid resulted in elevated PGE2 in the basolateral medium in a cyclooxygenase 1 (COX-1)-dependent manner. These data suggest that in the cortical collecting duct, locally produced and secreted PGE2 can stimulate ENaC-mediated transepithelial sodium transport.

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

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). 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. 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. 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.

The mTORC2‐SGK1 Signaling Module Mediates Local Sensing of Potassium in Kidney Tubule Principal Cells to Activate ENaC and Promote Potassium Secretion

ObjectivePotassium (K+) homeostasis requires precise regulation of K+ secretion by the kidney tubules. The serine‐threonine kinase, SGK1, is essential for normal responses to changes in extracellular K+ across the physiological range. SGK1 stimulates K+ secretion predominantly by activating the epithelial sodium channel (ENaC), which enhances electrogenic Na+ transport into K+ secretory cells and increases the electrical driving force for K+ secretion. Although it is well established that SGK1 activity is regulated through phosphorylation by the kinase, mTOR complex 2 (mTORC2), the physiologically relevant feedback systems that control this phosphorylation remain poorly characterized. In this study, we identified a direct effect of K+ in cortical collecting duct principal cells to stimulate ENaC and enhance K+ secretion, and set out to characterize the underlying mechanism.MethodsmpkCCD cortical collecting duct (CCD) cells were grown on Transwell filters and electrical properties were measured by Evometer following changes in extracellular [K+]. Following electrophysiological measurements, cells were harvested and processed for immunoblot analysis. Additional experiments were performed using patch clamp to detect ENaC currents in intact CCD isolated from mice and subjected to acute changes in bath [K+]. SGK1 phosphorylation state in response to changes in [K+] were also assessed in wild type and WNK1 deficient HEK‐293 cells.ResultsUsing whole cell patch clamp ex vivo, we found that in intact, isolated mouse collecting duct, a change in extracellular [K+] rapidly alters ENaC activity. Further, in cultured collecting duct cells an increase in basolateral but not apical [K+] led to enhanced ENaC‐dependent apical Na+ transport which paralleled an increase in the activated form of SGK1 (phosphorylated at S422). Pharmacologic inhibition of SGK1 or mTOR blocked the K+‐dependent activation of ENaC. Interestingly, the K+‐induced increase in SGK1 phosphorylation was not accompanied by an increase in phosphorylation of Akt2, a close relative of SGK1, which is also phosphorylated by mTORC2. We further explored the underlying mechanism for specific SGK1 activation by testing SGK1 and Akt2 phosphorylation in HEK‐293 cells lacking WNK1 (through CRISPR‐mediated gene deletion). Loss of WNK1 markedly impaired K+‐stimulated phosphorylation of SGK1 but not of Akt2. Additionally, we found that mutation of the chloride binding site of WNK1 led to the loss of K regulation of SGK1 phosphorylation. Further, in cells incubated in low Cl‐medium, increased extracellular K+ failed to increase SGK1 phosphorylation.ConclusionThese data strongly suggest that extracellular K+ can modulate mTORC2‐dependent activation of SGK1 through effects on Cl‐ binding to WNK1, leading to activation of ENaC and increased K+ secretion. We propose a model in which this signaling system supports a cell‐autonomous feedback loop within principal cells, which plays a key role in K+ homeostasis.Support or Funding InformationR01‐DK56695 to DP, R01‐DK54983 to WHW, the James Hilton Manning and Emma Austin Manning Foundation to DP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A new coupling of an acid-base transporter to PKD and cyst formation.

A new coupling of an acid-base transporter to PKD and cyst formation.

Cyclosporin A Induces Hypertension via a Cholesterol‐ and ENaC‐Dependent Mechanism

Hypertension related to the use of immunosuppressive drugs, particularly cyclosporin A (CsA), presents one of the major problems in the management of transplant recipients. Our recent studies suggest a hypothesis that CsA may cause hypertension by stimulating the renal epithelial sodium channel (ENaC) via a pathway associated with elevated intracellular cholesterol (Cho). To further determine the mechanism, we performed confocal microscopy and patch‐clamp experiments using cultured cortical collecting duct (CCD) principal cells and measured the blood pressure in mice. Confocal data show that CsA and Cho increased both the length of microvilli of CCD cells where ENaC is located and the levels of phosphatidylinositol‐4,5‐bisphosphate (PIP2) in microvilli. Conversely, inhibition of Cho synthesis with lovastatin decreased the levels of PIP2, a well‐known ENaC activator. Patch‐clamp data shows that Cho increased ENaC activity in a PIP2‐dependent manner. Interestingly, CsA elevated the blood pressure in mice and the elevation was abolished by either inhibition of Cho synthesis with lovastatin or blockade of ENaC with amiloride. These results suggest that CsA increases the blood pressure of mice by stimulating ENaC via a pathway associated with the elevation of intracellular Cho and PIP2 in the principal cells of CCD. However, it remains to be determined whether statins or amiloride can be used for the treatment of CsA‐induced hypertension.Support or Funding Information(Supported by NIH R01DK100582 to H.M.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells.

Exosomes are endosome-derived nanovesicles that are involved in cellular communication and signaling. Exosomes are produced by epithelial cells and are found in biologic fluids including blood and urine. The packaged material within exosomes includes proteins and lipids, but the molecular comparison within exosome subtypes is largely unknown. The purpose of this study was to investigate differences between exosomes derived from the apical plasma membrane and basolateral plasma membrane of polarized murine cortical collecting duct principal cells. Nanoparticle tracking analysis showed that the size and concentration of apical and basolateral exosomes remained relatively stable across 3 different temperatures (23, 37, and 42°C). Liquid chromatography-tandem mass spectrometry analysis revealed marked differences between the proteins packaged within the two types of exosomes from the same cells. Several proteins expressed at the inner leaflet of the plasma membrane, including α-actinin-1, moesin, 14-3-3 protein ζ/δ, annexin A1/A3/A4/A5/A6, clathrin heavy chain 1, glyceraldehyde-3-phosphate dehydrogenase, α-enolase, filamin-A, and heat shock protein 90, were identified in samples of apical plasma membrane-derived exosomes, but not in basolateral plasma membrane exosomes from mouse cortical collecting duct cells. In addition to differences at the protein level, mass spectrometry-based shotgun lipidomics analysis showed significant differences in the lipid classes and fatty acid composition of the two types of exosomes. We found higher levels of sphingomyelin and lower levels of cardiolipin, among other phospholipids in the apical plasma membrane compared to the basolateral plasma membrane exosomes. The molecular analyses of exosome subtypes presented herein will contribute to our understanding of exosome biogenesis, and the results may have potential implications for biomarker discovery.-Dang, V. D., Jella, K. K., Ragheb, R. R. T., Denslow, N. D., Alli, A. A. Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells.

ER and Mitochondria Regulate Intracellular Ca2+ and ENaC in Mouse Renal CCD Principal Cells

Cortical collecting duct (CCD) principal cells fine tune total body salt and water balance. Part of this regulation is via intracellular Ca2+; in particular, intracellular Ca2+ regulates renal epithelial sodium channels (ENaC). We have shown that mitochondria in principal cells sequester intracellular Ca2+ to form microdomains within cells with different Ca2+ concentrations in different areas of the cell, thereby allowing differential regulation of ENaC in different parts of the cell. Failure to establish the microdomains produces disorders of Ca2+ signaling including hypertension and polycystic kidney disease. Therefore, determining how mitochondria take up Ca2+ to form these domains is crucial to understanding pathophysiology. Mitochondria in CCD principal cells interact with the ER via voltage‐dependent anion channel 1 (VDAC1) on the outer mitochondrial membrane and IP3 receptor subtype 3 (IP3RIII) on the ER membrane to form a signaling complex known as mitochondrial‐associated ER membranes (MAMs). We hypothesize that MAMs are important for mitochondrial Ca2+ sequestration by allowing the rapid release of Ca2+ from ER into the cytosol to produce a local increase in intracellular Ca2+. At the same time the MAM complex activates mitochondrial VDAC1 to take up Ca2+ and prevent a persistent or global increase in Ca2+. Subsequently, mitochondria slowly return sequestered Ca2+ to the ER to restore ER Ca2+ stores. To determine whether the absence of VDAC1 disrupts MAMs, we perfused the kidneys of VDAC1 knockout mice and their littermate wild‐type mice with glutaraldehyde. TEM imaging indicates that although MAMs in the CCD principal cells were intact in both VDAC1 knockout and wild type mice, the mitochondria of the VDAC1 knockouts were larger than those of their wild‐type counterparts. To examine whether MAMs affect Ca2+ signaling domains, we imaged mpkCCD cells (n = 5) transfected with green Lck‐GCaMP2 (AddGene), a membrane‐associated Ca2+ sensor, and treated the cells with MDIVI‐1, an agent that selectively inhibits MAMs by blocking the dynamin‐dependent processes required for MAM formation. We applied UTP apically to increase intracellular Ca2+ beneath the apical membrane and found that Ca2+ increased strongly near the apical membrane, but only weakly in other parts of the cell. In cells treated with MDIVI‐1, Ca2+ increased less but was more uniformly distributed in all parts of the cell. To test whether VDAC1 influences ENaC activity in vivo, since ENaC currents increase as intracellular Ca2+ decreases, we examined ENaC regulation in renal tubules from VDAC1 knockout mice compared to wildtype mice on diets with different amounts of salt. We dissected and split open the cortical collecting duct, and then applied single‐channel patch clamp to the CCD principal cells. The activity of ENaC in wildtype mice decreased with increased sodium in the diet. ENaC activity in VDAC1 was significantly higher than wild type mice and activity did not depend upon dietary sodium. We conclude that inhibition of the associations between mitochondria and ER interferes with local Ca2+ signaling and therefore disrupts the ability of Ca2+ to regulate ENaC.Support or Funding InformationSupported by APS UGSRF and Emory's Robert P. Apkarian Integrated Electron Microscopy Core. VDAC1 knockout mice were obtained from Dr. William Craigen at Baylor University.

The Presence of Mitochondrial Associated ER Membranes (MAM) in Cortical Collecting Duct Principal Cells

Mitochondria have an established role as the ‘powerhouse of the cell’; additionally, they have been recently implicated in the formation of calcium signaling microdomains. In most epithelia, mitochondria can associate directly with IP3 receptors on the endoplasmic reticulum forming mitochondria‐associated ER membranes or MAMs. This interaction is facilitated by the tethering of the voltage dependent anion channel (VDAC) in the outer mitochondrial membrane and the IP3RIII on the ER. Ca2+ can move directly through the IP3RIII into the mitochondria via the VDAC1 channel. Interestingly, IP3RIII is the same isoform of IP3R known to mediate the inhibition of the epithelial sodium channel (ENaC) downstream of Gq proteins. Based on the knowledge that IP3R and mitochondrial Ca2+ both regulate ENaC activity, we hypothesized that MAMs exist in the cortical collecting duct (CCD) cells and are involved in the regulation of ENaC. MpkCCD cells were stained with mitotrackerRED and ERtrackerBlue and imaged with confocal microscopy, super resolution microscopy and we found that there is a strong association between both organelles. TEM images for mouse principal cells were obtained and further confirmed the association between ER and mitochondria. Wild type and VDAC1 kidneys were stained with fluorescently tagged VDAC1 and IP3R antibodies against these proteins. We found that the proteins co‐localize in kidney tissue and VDAC1 is present in mitochondrial membranes (as expected), but also in the plasma membrane of the CCD principal cells. Because of the ionic conditions across the outer mitochondrial membranes and across the plasma membrane, only VDAC1 in the mitochondria are likely to be active pathways for the movement of calcium. We concluded that mitochondria and ER associate in kidney cortical collecting duct principal cells through VDAC1 and IP3R complexes. In the future we will perform experiments to test whether MAMs are involved in the regulation of ENaC and their underlying mechanisms.Support or Funding InformationThis work was supported by APS Summer Undergraduate Research Programs to LG‐P and by NIH R01‐DK100582 to HPM

Data integration in physiology using Bayes' rule and minimum Bayes' factors: deubiquitylating enzymes in the renal collecting duct.

A major challenge in physiology is to exploit the many large-scale data sets available from "-omic" studies to seek answers to key physiological questions. In previous studies, Bayes' theorem has been used for this purpose. This approach requires a means to map continuously distributed experimental data to probabilities (likelihood values) to derive posterior probabilities from the combination of prior probabilities and new data. Here, we introduce the use of minimum Bayes' factors for this purpose and illustrate the approach by addressing a physiological question, "Which deubiquitylating enzymes (DUBs) encoded by mammalian genomes are most likely to regulate plasma membrane transport processes in renal cortical collecting duct principal cells?" To do this, we have created a comprehensive online database of 110 DUBs present in the mammalian genome (https://hpcwebapps.cit.nih.gov/ESBL/Database/DUBs/). We used Bayes' theorem to integrate available information from large-scale data sets derived from proteomic and transcriptomic studies of renal collecting duct cells to rank the 110 known DUBs with regard to likelihood of interacting with and regulating transport processes. The top-ranked DUBs were OTUB1, USP14, PSMD7, PSMD14, USP7, USP9X, OTUD4, USP10, and UCHL5. Among these USP7, USP9X, OTUD4, and USP10 are known to be involved in endosomal trafficking and have potential roles in endosomal recycling of plasma membrane proteins in the mammalian cortical collecting duct.

Interleukin-1β as a driver of renal NGAL production

Neutrophil gelatinase-associated lipocalin (NGAL) is increasingly regarded as a biomarker of acute kidney injury, or kidney injury in general, but the stimuli responsible for its production are incompletely understood. This study tested the relationship between the pro-inflammatory cytokine interleukin-1β (IL-1β) and both circulating and renal NGAL, using chronic subcutaneous infusion of IL-1β in mice and tissue culture of renal cell lines. Following a 14-day subcutaneous infusion of vehicle or IL-1β (10ng/h) in male C57Bl/6 mice, a striking positive correlation (r2=0.94; P<0.01) was observed between plasma IL-1β and NGAL concentrations. NGAL was markedly increased in the kidneys of IL-1β-infused mice compared with vehicle-treated mice, both at the protein and mRNA level, indicating increased local as well as systemic production of NGAL. Immunohistochemical staining revealed prominent increases of NGAL in the proximal tubular epithelium of IL-1β infused mice. These effects occurred in the absence of overt renal injury, with plasma creatinine concentration not significantly different between groups. Further showing that IL-1β has a direct effect on NGAL production by tubular epithelial cells, exposure of a proximal tubular cell line (HK-2 cells) and a cortical collecting duct principal cell line (mpkCCD cells) to IL-1β for 24h produced a significant increase of NGAL mRNA levels (>30-fold). These data indicate IL-1β serves as a powerful stimulus for renal production of NGAL.

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.

Expression of a Diverse Array of Ca2+-Activated K+ Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney.

The voltage- and Ca2+-activated, large conductance K+ channel (BK, maxi-K) is expressed in the collecting duct system of kidney where it underlies flow- and Ca2+-dependent K+ excretion. To determine if other Ca2+-activated K+ channels (KCa) may participate in this process, mouse kidney and the K+-secreting mouse cortical collecting duct (CCD) cell line, mCCDcl1, were assessed for TRPV4 and KCa channel expression and cross-talk. qPCR mRNA analysis and immunocytochemical staining demonstrated TRPV4 and KCa expression in mCCDcl1 cells and kidney connecting tubule (CNT) and CCD. Three subfamilies of KCa channels were revealed: the high Ca2+-binding affinity small-conductance SK channels, SK1and SK3, the intermediate conductance channel, IK1, and the low Ca2+-binding affinity, BK channel (BKα subunit). Apparent expression levels varied in CNT/CCD where analysis of CCD principal cells (PC) and intercalated cells (IC) demonstrated differential staining: SK1:PC<IC, and SK3:PC>IC, IK1:PC>IC, BKα:PC = IC, and TRPV4:PC>IC. Patch clamp analysis and fluorescence Ca2+ imaging of mCCDcl1 cells demonstrated potent TRPV4-mediated Ca2+ entry and strong functional cross-talk between TRPV4 and KCa channels. TRPV4-mediated Ca2+ influx activated each KCa channel, as evidenced by selective inhibition of KCa channels, with each active KCa channel enhancing Ca2+ entry (due to membrane hyperpolarization). Transepithelial electrical resistance (TEER) analysis of confluent mCCDcl1 cells grown on permeable supports further demonstrated this cross-talk where TRPV4 activation induce a decrease in TEER which was partially restored upon selective inhibition of each KCa channel. It is concluded that SK1/SK3 and IK1 are highly expressed along with BKα in CNT and CCD and are closely coupled to TRPV4 activation as observed in mCCDcl1 cells. The data support a model in CNT/CCD segments where strong cross talk between TRPV4-mediated Ca2+ influx and each KCa channel leads to enhance Ca2+ entry which will support activation of the low Ca2+-binding affinity BK channel to promote BK-mediated K+ secretion.

ENaC in Renal Cortical Collecting Duct Principal Cells Is Stimulated by Increasing Intracellular Ca2+ in The Basal Compartment of The Cell Via a Process Involving Src but not CAMK II

We have recently shown that intracellular Ca2+ ([Ca2+]i) in renal cortical collecting duct (CCD) principal cells is compartmentalized and that this compartmentalization is maintained by mitochondria which localize in two separate areas beneath the apical and basolateral membranes and sequester Ca2+ to greatly reduce the rate of cytosolic Ca2+ diffusion. When [Ca2+]i is increased in the cytosolic space adjacent to the apical plasma membrane, ENaC is inhibited, but when [Ca2+]i is increased in the cytosolic space adjacent to the basolateral membrane, ENaC activity is stimulated. While the pathways mediating the effects of apical [Ca2+]i are well characterized, the pathways that transduce the basolateral pathways are unknown. We tested which signaling molecules may be mediating these effects. We used a combination of transepithelial current measurements and single channel patch clamping to identify these molecules. Inhibitors of common Ca2+ ‐sensitive signaling molecules were used in combination with either ionomycin or methyl‐thio‐ATP applied basolaterally to increase [Ca2+]i. Since some basolaterally expressed receptor tyrosine kinases stimulate ENaC via activation of Src and since Src is a Ca2+ ‐sensitive protein, we first tested whether basolateral [Ca2+]i stimulates ENaC via a Src‐mediated pathway. Basolaterally applied ionomycin (15 μM) or methyl‐thio‐ATP (50 μM), causes a two‐fold increase in ENaC open probability (N≥6; P&lt;0.05 per group), but has no effect in the presence of the Src inhibitor PP2 (12 μM; N=6; P&gt;0.5). We also considered another Ca2+ ‐dependent that might have stimulated ENaC. Hypotonicity can stimulate ENaC via activation of calmodulin‐dependent protein kinase II (CAMK II). Using concentrations of the CAMKII inhibitor KN93 from 1–100μM, we found no effect of this drug on amiloride‐sensitive transepithelial current induced by basolateral [Ca2+]i. Overall, we conclude that Src mediates the stimulatory effects of basolateral [Ca2+]i on ENaC but that CAMKII likely does not. Whether this occurs via cross talk with receptor tyrosine kinase signaling pathways remains to be seen.Support or Funding InformationNIH R37‐DK037963 to DCE, NIH R01‐DK100582 to HM and AHA 13POST16820072 to TLT

TRPM4 Is Expressed in and Carries a Nonselective Cation Current Through the Apical Membrane of Mouse Cortical Collecting Duct Principal Cells

Maintaining daily water, K+‐ and Na+‐balance is an important function of principal cells of the mouse cortical collecting duct (CCD). Under physiological conditions, these cells express a Ca2+ ‐activated nonselective cation channel (NSCCa), the molecular identity of which has been unknown. This study sought to identify whether the nonselective cation channel, TRPM4, functions as a NSCCa in principal cells of mouse cortical collecting duct.Using immunohistochemistry and western blotting, we show the presence of TRPM4 channel protein in the mouse cortical collecting duct cell line mpkCCDc14. Single channel recordings showed a current that is carried by monovalent cations and is Ca2+ sensitive and exhibits equal permeability for Na+ and K+. The corresponding current–voltage relationship of this channel was linear with a slope conductance 23.4 pS, which is the same as that observed from heterologously expressed TRPM4 channels. TRPM4 is inhibited by ATP and stimulated by PIP2. Application of 1 mM ATP to the “cytoplasmic” bath solution of an inside‐out patch almost completely inhibited channel activity but washing out ATP fully restored the channel activity. The channel activity significantly increased after adding 1 μM PI(4,5)P2 to the “cytoplasmic” bath solution. The TRPM4 inhibitor 9‐phenanthrol (100 μM) strongly inhibited the endogenous current. These biophysical properties of NSCCa are identical to those described for TRPM4. Acute application of H2O2 (100 μM and 500 μM) was unable to stimulate TRPM4 activity inside‐out patches; however, using a cell surface biotinylation assay, we showed that treating the mpkCCDc14 with 100 μM H2O2 for 24hrs significantly decreased cell surface levels of TRPM4 protein.Overall, we conclude that TRPM4 is the molecular identity of NSCCa in mpkCCDc14. Further study will be needed to determine the physiological relevance of this channel.Support or Funding InformationNIH R01‐DK100582 to HM

Insulin and IGF-1 activate Kir4.1/5.1 channels in cortical collecting duct principal cells to control basolateral membrane voltage.

Potassium Kir4.1/5.1 channels are abundantly expressed at the basolateral membrane of principal cells in the cortical collecting duct (CCD), where they are thought to modulate transport rates by controlling transepithelial voltage. Insulin and insulin-like growth factor-1 (IGF-1) stimulate apically localized epithelial sodium channels (ENaC) to augment sodium reabsorption in the CCD. However, little is known about their actions on potassium channels localized at the basolateral membrane. In this study, we implemented patch-clamp analysis in freshly isolated murine CCD to assess the effect of these hormones on Kir4.1/5.1 at both single channel and cellular levels. We demonstrated that K(+)-selective conductance via Kir4.1/5.1 is the major contributor to the macroscopic current recorded from the basolateral side in principal cells. Acute treatment with 10 μM amiloride (ENaC blocker), 100 nM tertiapin-Q (TPNQ; ROMK inhibitor), and 100 μM ouabain (Na(+)-K(+)-ATPase blocker) failed to produce a measurable effect on the macroscopic current. In contrast, Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 μM), virtually abolished whole cell K(+)-selective conductance. Insulin (100 nM) markedly increased the open probability of Kir4.1/5.1 and nortriptyline-sensitive whole cell current, leading to significant hyperpolarization of the basolateral membrane. Inhibition of the phosphatidylinositol 3-kinase cascade with LY294002 (20 μM) abolished action of insulin on Kir4.1/5.1. IGF-1 had similar stimulatory actions on Kir4.1/5.1-mediated conductance only when applied at a higher (500 nM) concentration and was ineffective at 100 nM. We concluded that both insulin and, to a lesser extent, IGF-1 activate Kir4.1/5.1 channel activity and open probability to hyperpolarize the basolateral membrane, thereby facilitating Na(+) reabsorption in the CCD.

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.

Cross-talk between insulin and IGF-1 receptors in the cortical collecting duct principal cells: implication for ENaC-mediated Na+ reabsorption.

Insulin and IGF-1 are recognized as powerful regulators of the epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron. As previously described, these hormones both acutely increase ENaC activity in freshly isolated split open tubules and cultured principal cortical collecting duct cells. The present study was aimed at differentiating the effects of insulin and IGF-1 on Na+ transport in immortalized mpkCCDcl4 cells and defining their interrelations. We have shown that both insulin and IGF-1 applied basolaterally, but not apically, enhanced transepithelial Na+ transport in the mpkCCDcl4 cell line with EC50 values of 8.8 and 14.5 nM, respectively. Insulin treatment evoked phosphorylation of both insulin and IGF-1 receptors, whereas the effects of IGF-1 were more profound on its own receptor rather than the insulin receptor. AG-1024 and PPP, inhibitors of IGF-1 and insulin receptor tyrosine kinase activity, diminished insulin- and IGF-1-stimulated Na+ transport in mpkCCDcl4 cells. The effects of insulin and IGF-1 on ENaC-mediated currents were found to be additive, with insulin likely stimulating both IGF-1 and insulin receptors. We hypothesize that insulin activates IGF-1 receptors in addition to its own receptors, making the effects of these hormones interconnected.

Role of Rho GDP Dissociation Inhibitor α in Control of Epithelial Sodium Channel (ENaC)-mediated Sodium Reabsorption

The epithelial sodium channel (ENaC) is expressed in the aldosterone-sensitive distal nephron where it performs sodium reabsorption from the lumen. We have recently shown that ENaC activity contributes to the development of salt-induced hypertension as a result of deficiency of EGF level. Previous studies revealed that Rho GDP-dissociation inhibitor α (RhoGDIα) is involved in the control of salt-sensitive hypertension and renal injury via Rac1, which is one of the small GTPases activating ENaC. Here we investigated the intracellular mechanism mediating the involvement of the RhoGDIα/Rac1 axis in the control of ENaC and the effect of EGF on ENaC in this pathway. We demonstrated that RhoGDIα is highly expressed in the cortical collecting ducts of mice and rats, and its expression is down-regulated in Dahl salt-sensitive rats fed a high salt diet. Knockdown of RhoGDIα in cultured cortical collecting duct principal cells increased ENaC subunits expression and ENaC-mediated sodium reabsorption. Furthermore, RhoGDIα deficiency causes enhanced response to EGF treatment. Patch clamp analysis reveals that RhoGDIα significantly decreases ENaC current density and prevents its up-regulation by RhoA and Rac1. Inhibition of Rho kinase with Y27632 had no effects on ENaC response to EGF either in control or RhoGDIα knocked down cells. However, EGF treatment increased levels of active Rac1, which was further enhanced in RhoGDIα-deficient cells. We conclude that changes in the RhoGDIα-dependent pathway have a permissive role in the Rac1-mediated enhancement of ENaC activity observed in salt-induced hypertension.

Cholesterol inhibits ROMK channels by holding PI(4,5)P2 in microvilli (1181.17)

The kidney plays an important role in maintaining ion balance in the blood. K+ balance is tightly regulated by a K+ channel called ROMK1. We used cultured mouse cortical collecting duct principal cells (mpkCCDc14 line) to determine how ROMK1 is regulated by cholesterol. Previous studies suggest that cholesterol inhibits ROMK channels via a direct mechanism. Surprisingly, our data demonstrate that ROMK1 channels are not located in cholesterol‐rich lipid rafts in cultured CCD cells. Patch‐clamp data demonstrated that a small‐conductance K+ (ROMK1) channel in mpkCCDc14 cells was inhibited by cholesterol and activated by extraction of cholesterol with methyl‐beta‐cyclodextrin (MbetaCD). However, confocal microscopy data showed that ROMK1 was not in the microvilli where cholesterol‐rich lipid rafts are located, but in the planar region of the apical membrane of mpkCCDc14 cells, and that phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P2), a well‐known activator of ROMK channels, was found only in the microvilli under resting conditions. Interestingly, after cholesterol synthesis was inhibited by lovastatin, PI(4,5)P2 diffused into planar region with a consequence of activation of ROMK channels. Activation of ROMK1 channels by MbetaCD was reversed by addition of exogenous cholesterol, but the activation did not occur when PI(4,5)P2 was sequestered with its antibody. These results suggest that cholesterol inhibits ROMK1 channels, at least in part, by limiting PI(4,5)P2 diffusion from microvilli to planar region of the renal CCD cell apical membrane. Since we have reported that cyclosporine A (CsA), an immunosuppressant drug which causes hyperkalemia, can alter the levels of cholesterol in renal epithelial cells, this study provides a basis for researchers to determine the mechanism of CsA‐induced hyperkalemia.Grant Funding Source: supported by NIH 5R01‐DK067110 to HPM and by NSF of China 81130028 to B‐ZS