Channel-inducing Factor Research Articles

Abstract 046: Deep Sequencing in mpkCCD Collecting Duct Cells Identifies Transcriptional Response to Vasopressin

Blood pressure regulation depends in part on control of ion and water transport in the renal collecting duct by vasopressin and other hormones. To identify genes that are transcriptionally regulated by vasopressin in cultured mouse collecting duct cells (mpkCCD), we carried out both RNA-seq (n=9) and ChIP-seq for RNA polymerase II (Pol-II, n=3) after 24-hr treatment with vasopressin analog dDAVP or vehicle (Illumina HiSeq2000 sequencer). RNA-seq analysis showed significant changes in 33 transcripts out of 8393 quantified (Volcano plot thresholds: p<0.05 (Wilcoxon) and log2[ratio] outside of 99% confidence interval (CI) for vehicle-vs-vehicle controls). More transcripts increased (26) than decreased (7). The largest increases were seen for the transcriptional coactivator Cited1 (log2[dDAVP/Vehicle]= 5.2) and the water channel Aqp2 (log2[dDAVP/Vehicle]= 4.2). The ChIP-seq analysis showed significantly increased RNA-Pol-II binding in 209 genes versus only 9 with decreases. Plotting RNA-seq data versus ChIP-seq data for 3883 genes revealed 43 genes that exceeded the 95% CI for control-vs-control measurements for both variables, i.e., are transcriptionally regulated. The transcriptionally regulated genes included those involved in control of Na+/Cl- transport and/or blood pressure, viz. Fxyd4 (aldosterone-regulated channel-inducing factor), Tsc22d3 (the aldosterone-regulated leucine-zipper protein GILZ), Atp1b1 (Na+-K+-ATPase beta subunit) and Sik1 (salt-inducible kinase 1). Among transcriptionally upregulated genes were several transcription factors ( Elf3 , Nfkbiz , Nr4a1 , Myc ) that form the core of a proposed vasopressin/cAMP-activated transcriptional network. Conclusions: 1) Vasopressin signaling effects on transcription are biased with more genes increased than decreased. 2) Among the genes that are transcriptionally regulated in response to vasopressin are several that have been implicated in regulation of salt and water transport in the collecting duct.

Structural studies of apoptosis and ion transport regulatory proteins in membranes.

Solid-state NMR spectroscopy is being used to determine the structures of membrane proteins involved in the regulation of apoptosis and ion transport. The Bcl-2 family includes pro- and anti-apoptotic proteins that play a major regulatory role in mitochondrion-dependent apoptosis or programmed cell death. The NMR data obtained for (15)N-labeled anti-apoptotic Bcl-xL in lipid bilayers are consistent with membrane association through insertion of the two central hydrophobic alpha-helices that are also required for channel formation and cytoprotective activity. The FXYD family proteins regulate ion flux across membranes, through interaction with the Na(+), K(+)-ATPase, in tissues that perform fluid and solute transport or that are electrically excitable. We have expressed and purified three FXYD family members, Mat8 (mammary tumor protein), CHIF (channel-inducing factor) and PLM (phospholemman), for structure determination by NMR in lipids. The solid-state NMR spectra of Bcl-2 and FXYD proteins, in uniaxially oriented lipid bilayers, give the first view of their membrane-associated architectures.

Dissecting mineralocorticoid receptor structure and function

The molecular mechanisms by which aldosterone regulates epithelial sodium transport in the distal colon and the distal nephron remain to be fully elucidated. Aldosterone acts via the mineralocorticoid receptor (MR) to induce the expression of genes whose products are involved in sodium transport. The structural basis of MR interactions with aldosterone has been examined by creating chimeras of the MR and the closely related glucocorticoid receptor; we have exploited differences in ligand-binding specificity to determine the region(s) of the MR that confer aldosterone-binding specificity. These findings have been related to a three-dimensional model of the MR based on the crystal structure of the progesterone receptor. These studies have been extended to include the characterisation of interactions between the N- and C-termini of the MR. We have characterised six genes that are regulated in vivo in the distal colon and/or kidney of the rat that are directly and acutely regulated by aldosterone administration: the three subunits of the epithelial sodium channel, serum and glucocorticoid-induced kinase, channel-inducing factor and K-ras2A. These studies provide insights into the molecular pathways that mediate aldosterone-induced amiloride-sensitive epithelial sodium transport.

Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins

The preparation of oriented, hydration-optimized lipid bilayer samples, for NMR structure determination of membrane proteins, is described. The samples consist of planar phospholipid bilayers, containing membrane proteins, that are oriented on single pairs of glass slides, and are placed in the coil of the NMR probe with the bilayer plane perpendicular to the direction of the magnetic field. Lipid bilayers provide a medium that closely resembles the biological membrane, and sample orientation both preserves the intrinsic membrane-defined directional quality of membrane proteins, and provides the mechanism for resonance line narrowing. The hydration-optimized samples overcome some of the difficulties associated with multi-dimensional, high-resolution, solid-state NMR spectroscopy of membrane proteins. These samples have greater stability over the course of multi-dimensional NMR experiments, they have lower sample conductance for greater rf power efficiency, and enable greater rf coil filling factors to be obtained for improved experimental sensitivity. Sample preparation is illustrated for the membrane protein CHIF (channel inducing factor), a member of the FXYD family of ion transport regulators.

Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers

The proteins PLM (phospholemman), CHIF (channel inducing factor), and Mat8 (mammary tumor protein 8 kDa) are members of the FXYD family of ion transport regulatory membrane proteins. Here we describe their cloning and expression in Escherichia coli, and their purification for NMR structural studies in lipid micelles and lipid bilayers. The molecular masses of the purified recombinant FXYD proteins, determined from SDS-PAGE and from MALDI TOF mass spectrometry, reflect monomeric species. The solution NMR and CD spectra in SDS micelles show that they adopt helical conformations. The solid-state NMR spectra in lipid bilayers give the first view of their transmembrane architecture.

Colonic H(+)-K(+)-ATPase in K(+) conservation and electrogenic Na(+) absorption during Na(+) restriction.

Upregulation of the colonic H(+)-K(+)- ATPase (cHKA) during hyperaldosteronism suggests that it functions in both K(+) conservation and electrogenic Na(+) absorption in the colon when Na(+)-conserving mechanisms are activated. To test this hypothesis, wild-type (cHKA(+/+)) and cHKA-deficient (cHKA(-/-)) mice were fed Na(+)-replete and Na(+)-restricted diets and their responses were analyzed. In both genotypes, Na(+) restriction led to reduced plasma Na(+) and increased serum aldosterone, and mRNAs for the epithelial Na(+) channel (ENaC) beta- and gamma-subunits, channel-inducing factor, and cHKA were increased in distal colon. Relative to wild-type controls, cHKA(-/-) mice on a Na(+)-replete diet had elevated fecal K(+) excretion. Dietary Na(+) restriction led to increased K(+) excretion in knockout but not in wild-type mice. The amiloride-sensitive, ENaC-mediated short-circuit current in distal colon was significantly reduced in knockout mice maintained on either the Na(+)-replete or Na(+)-restricted diet. These results demonstrate that cHKA plays an important role in K(+) conservation during dietary Na(+) restriction and suggest that cHKA-mediated K(+) recycling across the apical membrane is required for maximum electrogenic Na(+) absorption.

Regulation of ROMK and channel-inducing factor (CHIF) in acute renal failure due to ischemic reperfusion injury

Acute renal failure caused by ischemia followed by reperfusion is often associated with severe hyperkalemia. The present study was undertaken to characterize the effects of renal ischemia and reperfusion on plasma potassium (K) and on the gene expression of channel-inducing factor (CHIF), a putative K channel regulator, and of ROMK, the distal nephron secretory K channel. The following groups of rats were studied: (1) sham operated (sham); (2) after one hour of ischemia by bilateral renal artery clamping (I), and after one hour of ischemia; (3) one hour of reperfusion (I-R 1 h); (4) 24 hours of reperfusion (I-R 24 h); (5) 48 hours of reperfusion (I-R 48 h); and (6) 72 hours reperfusion (I-R 72 h). The expression of CHIF and ROMK was examined by Northern blot hybridization in renal cortex, medulla, and papilla and in the colon. The abundance of ROMK protein was determined in the renal cortex and medulla by immunoblotting. Maximal plasma creatinine and potassium levels after ischemia and reperfusion were 470 +/- 16 micromol/L, P < 0.0001 versus sham, and 9.65 +/- 0.33 mmol/L, P < 0.0001 versus sham, respectively. The expression of CHIF was significantly down-regulated in the medulla and papilla, with a maximal decrease of 80% at 48 to 72 hours. In contrast, a most significant increase in CHIF mRNA expression (250% of baseline) was noted in the colon after 24 to 48 hours of reperfusion. ROMK expression was reduced in the cortex and was completely abolished in the medulla at 48 to 72 hours of reperfusion. Ischemia and reperfusion injury significantly decreased ROMK protein abundance to 10% of control in the medullary fractions. These results suggest that down-regulation of renal CHIF and ROMK may contribute at least partly to the hyperkalemia of acute renal failure after ischemia and reperfusion, while CHIF up-regulation in the colon may act as a compensatory mechanism of maintaining K balance via increased K secretion.

Possible molecular basis for changes in potassium handling in acute renal failure

Renal potassium excretion is diminished in acute renal failure (ARF). The gastrointestinal tract can compensate for deficient renal potassium excretion in many patients with ARF. For both impaired renal potassium excretion and gastrointestinal compensation in ARF, little is known about the role of potassium channels. We hypothesized that specific changes in the expression of the secretory renal outer medullary potassium channel (ROMK), and the potassium channel regulator, channel-inducing factor (CHIF), in kidney and colon could contribute to changes in potassium handling. Sprague-Dawley rats underwent uninephrectomy and 35 minutes of renal ischemia, followed by varying periods of reperfusion. Renal function, serum and urine potassium levels, and aldosterone levels were measured. The expression of messenger RNA (mRNA) for ROMK and CHIF in the kidney and CHIF in the colon were measured by Northern blot hybridization. Serum creatinine level was increased at 24 hours and started to decline at 48 hours of renal ischemia reperfusion injury (IRI). Serum potassium level was increased at 24 hours, further elevated at 48 hours of IRI, and returned to normal at 7 days of IRI. Urine potassium level was reduced at 24 and 48 hours. Northern blot analysis indicated that the expression of ROMK1 mRNA in the cortex or medulla remained unchanged at 24 hours but profoundly decreased (by 70% to 80%) at 48 hours (n = 4; P < 0.01). The expression of CHIF mRNA in the kidney cortex or medulla decreased by 25% to 30% at 24 hours and 35% to 40% at 48 hours of IRI (n = 4; P < 0.05 for each group). CHIF mRNA expression in the distal colon was moderately increased at 24 hours (approximately twofold) and significantly enhanced at 48 hours (more than threefold; P < 0.01; n = 4) of IRI. Serum aldosterone level was increased approximately threefold at 48 hours of IRI (P < 0.01; n = 6). In conclusion, (1) suppression of ROMK and CHIF in the kidney may contribute to decreased renal potassium excretion in ARF; (2) enhanced expression of CHIF in the distal colon in IRI could be an adaptive response to increase potassium excretion in the colon and help modify hyperkalemia in ARF; and (3) increased aldosterone levels, as a response to hyperkalemia, could be upregulating colonic CHIF.

Specificity in mineralocorticoid versus glucocorticoid action

The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) share considerable structural and functional homology. Overlapping effects on epithelial sodium transport are observed in vivo; in vitro, both are able to bind and transactivate through a common hormone response element. This has led several investigators to suggest that specificity is conferred primarily by prereceptor mechanisms, and we have addressed this question using both in vitro and in vivo approaches. Although the MR has been regarded as less transcriptionally active than the GR in vitro, significant differences are observed when epithelial rather than fibroblast cell lines are used. These differences are mediated by the N-termini of the receptors. Activation of intracellular signaling pathways differentially modulates MR- versus GR-mediated transactivation. Although these studies identify mechanisms by which specificity may be achieved, they do not prove that this occurs in vivo. Such studies have been limited by an absence of MR-regulated genes. Known candidate aldosterone-responsive genes have been examined in the rat distal colon; the time course and the specificity of the response to a single parenteral dose of corticosteroid has been characterized. The epithelial sodium channel beta and gamma subunit genes are both up-regulated within 60 minutes by either MR or GR activation. Similar responses are observed for the serum and glucocorticoid-regulated kinase and channel-inducing factor genes. All four genes show clear and rapid up-regulation of their mRNA levels by aldosterone, which is paralleled by GR-mediated up-regulation of expression. While they are indeed aldosterone-responsive genes, genes that are uniquely aldosterone-regulated remain to be identified.

Acute regulation by corticosteroids of channel-inducing factor gene messenger ribonucleic acid in the distal colon.

The molecular mechanisms by which corticosteroids affect fluid and electrolyte balance are unclear. Though glucocorticoid-responsive genes have been identified, genes regulated by aldosterone have not. CHIF (channel-inducing factor gene) is a recently identified gene that is up-regulated in the distal colon by chronic corticosteroid exposure, is expressed in the kidney, and induces a K+-specific current in Xenopus oocytes. The predicted protein shows similarity to gammaNa.K-ATPase, phospholemman, and Mat-8; all seem to be involved in ion transport. CHIF thus presents as a potential aldosterone target gene. In this study, CHIF expression was examined in rats in the acute timeframe of 0.5-4 h after corticosteroid administration. CHIF messenger RNA showed up-regulation by both mineralocorticoid and glucocorticoid receptor agonists in the distal colon, which was not diminished by cycloheximide. Corticosteroid regulation was not observed in the kidney. Basal and induced expression was absent in the lung and in all gastrointestinal tissues except colon, with expression increasing proximal to distal. CHIF is the first gene to show acute regulation by aldosterone and thus encodes a candidate aldosterone-induced protein. In addition, gammaNa.K-ATPase gene expression was found to be very low in colon and significantly higher in kidney. Regulation by corticosteroids was not evident in either tissue.

Acute Regulation by Corticosteroids of Channel-Inducing Factor Gene Messenger Ribonucleic Acid in the Distal Colon

Acute Regulation by Corticosteroids of Channel-Inducing Factor Gene Messenger Ribonucleic Acid in the Distal Colon

Differential regulation of CHIF mRNA by potassium intake and aldosterone.

The channel-inducing factor (CHIF) is an epithelial-specific transmembrane protein, which is induced by aldosterone in distal colon (but not in kidney) and can evoke K+ conductance in Xenopus oocytes. The current study examined the possibility that CHIF participates in maintaining K+ balance by assessing its regulation during variations in K+ intake. In adrenal-intact rats, high-K+ diet stimulated, whereas K+ deficiency downregulated, CHIF mRNA both in kidney and colon. The downregulation of CHIF observed in rats fed a low-K+ diet for different periods of time closely correlated with a decrease in plasma K+ but also with changes in aldosterone levels. To differentiate between the two, modulation of CHIF has been studied in adrenalectomized rats with and without corticosteroid supplementation. These experiments have demonstrated that a low-K+ intake suppresses CHIF mRNA, irrespective of aldosterone level. On the other hand, the upregulation evoked by a high-K+ load is apparent only in adrenal-intact rats. This is despite the fact that infusing rats with aldosterone and corticosterone does not increase the expression of this mRNA in kidney. These findings may suggest a role for CHIF in preserving K+ balance.

Cellular localization and regulation of CHIF in kidney and colon.

Channel inducing factor (CHIF) is a novel cDNA recently cloned from a rat distal colon cDNA library of dexamethasone-treated animals. While its expression in Xenopus oocytes evokes a potassium channel activity similar to that induced by Isk (minK), its cellular role is not clear. CHIF exhibits significant homologies with proteins that are putatively regulatory (phospholemman, gamma-subunit of Na(+)-K(+)-ATPase, Mat-8) while it differs from the small-conductance potassium channel Isk. We have studied the tissue specificity of CHIF expression in rat by in situ hybridization. CHIF is selectively present in the distal parts of the nephron (medullary and papillary collecting ducts and end portions of cortical collecting tubule) and in the epithelial cells of the distal colon. No expression of CHIF was found in renal proximal tubule, loop of Henle and distal tubule, proximal colon, small intestine, lung, choroid plexus, salivary glands, or brain. To gain some insight into CHIF function, we have investigated, using in situ hybridization and ribonuclease protection assay, whether CHIF mRNA expression could be altered in some situations. In the distal colon, corticosteroid hormones, sodium restriction, low-potassium diet, and metabolic acidosis significantly increased CHIF mRNA expression. In the kidney, metabolic acidosis was the only condition that showed an increase in CHIF mRNA expression. Some of these treatments also altered the expression of the colonic H(+)-K(+)-ATPase mRNA. In summary, CHIF mRNA is selectively expressed in the medullary collecting duct of the kidney and in the epithelium of the distal colon; its expression varies differently in these two target tissues after alterations in corticosteroid status, potassium depletion, and metabolic acidosis. The precise cell-specific functions of CHIF remain to be established.

A corticosteroid-induced gene expressing an "IsK-like" K+ channel activity in Xenopus oocytes.

Screening a rat colon cDNA library for aldosterone-induced genes resulted in the molecular cloning of a cDNA whose corresponding mRNA is strongly induced in the colon by dexamethasone, aldosterone, and a low NaCl diet. A similar mRNA was detected in kidney papilla but not in brain, heart, or skeletal muscle. Xenopus laevis oocytes injected with cRNA synthesized from this clone, designated CHIF (channel-inducing factor), express a K(+)-specific channel activity. The biophysical, pharmacological, and regulatory characteristics of this channel are very similar to those reported before for IsK (minK). These include: slow (tau > 20 s) activation by membrane depolarization with a threshold potential above -50 mV, blockade by clofilium, inhibition by phorbol ester, and activation by 8-bromoadenosine 3',5'-cyclic monophosphate and high cytoplasmic Ca2+. The primary structure of this clone, however, shows no homology to IsK. Instead, CHIF exhibits > 50% similarity to two other short bitopic membrane proteins, phospholemman and the gamma subunit of Na+K(+)-ATPase. The data are consistent with the possibility that CHIF is a member of a family of transmembrane regulators capable of activating endogenous oocyte transport proteins.