ROMK mRNA Research Articles

Cell‐autonomous expression of membrane proteins in kidney thick ascending limb epithelium is vasopressin‐dependent

The thick ascending limb (TAL) of the mammalian kidney exhibits functionally and structurally distinct patterns along its course from the border of the inner medulla to its transition to the distal convoluted tubule. While reabsorption of 25% of the filtered load is the principal task shared by all TAL cells, the transport of K+ and the divalent cations Ca++ and Mg++ is subject to axial and cellular heterogeneity and occurs along trans‐ or paracellular pathways. Differential expression of the renal outer medullary K+ channel (ROMK) has been associated with net K+ secretion or reabsorption, and claudin (Cldn) 10 and 16 with selective paracellular cation permeability. Earlier we had detected basolateral vasopressin (AVP) V2 receptor expression and substantial activating responsiveness of transport parameters along TAL upon adding AVP. Here we tested the hypothesis whether AVP modulates cell‐autonomous expression of membrane proteins involved in TAL epithelial ion transport.We analyzed perfusion‐fixed mouse and rat kidneys. Human kidney biopsies were studied for comparison. The effect of AVP was tested in AVP‐deficient Brattleboro rats using V2R‐specific desmopressin (dDAVP; 7 and 72 hours). NKCC2, ROMK, Kir4.1, Cldn‐10 and Cldn‐16 signals were analyzed using sensitive in situ hybridization, IHC, EM, and Western blot (WB).TAL morphological cell heterogeneity was obvious between kidney zones, but not at the cell‐to‐cell level within any zone. Morphologically heterogeneous “smooth” and “rough” patterns of neighboring TAL cells were not discovered, but instead, ROMK und Kir4.1 expression showed conspicuous heterogeneity in a mutually exclusive pattern in tubules of outer medulla and cortex of rodent and human kidneys. Most tight junctions of inner stripe TAL were Cldn‐10 positive. In its outermost layer, Cldn‐10‐ and ‐16 signals were co‐expressed; ROMK‐positive cells displayed Cldn‐10‐positive tight junctions between the adjacent ROMK‐positive cells whereas between ROMK‐negative cells, junctions were Cldn‐16‐positive. 72h dDAVP administration significantly increased the number of ROMK‐positive cells in outer medullary TAL. Kir4.1‐positive cells increased in parallel. Cldn‐10‐positive junctions increased at the expense of Cldn‐16‐positive junctions. WB showed 2.2‐fold increase for ROMK and 1.3‐fold for Kir4.1 (p<.05). Sensitive ISH revealed differential quantities of ROMK mRNA abundances in TAL, with increases in the number of cells expressing ROMK mRNA strongly upon dDAVP.These results demonstrate cell‐autonomous regulation of crucial TAL cell membrane proteins in response to activating stimuli in the absence of major cell morphological changes. Variable expression of ROMK, Kir4.1, and Cldn‐10 and ‐16 signals likely reflected vasopressindependent adaptive features in renal solute handling in the renal outer medulla.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial ROMK Channel Is a Molecular Component of MitoK ATP

Activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) has been implicated in the mechanism of cardiac ischemic preconditioning, yet its molecular composition is unknown. To use an unbiased proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K(+) channel underlying mitoK(ATP). Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and ROMK mRNA was confirmed to be present in neonatal rat ventricular myocytes and adult hearts. ROMK2, a short form of the channel, is shown to contain an N-terminal mitochondrial targeting signal, and a full-length epitope-tagged ROMK2 colocalizes with mitochondrial ATP synthase β. The high-affinity ROMK toxin, tertiapin Q, inhibits mitoK(ATP) activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, short hairpin RNA-mediated knockdown of ROMK inhibits the ATP-sensitive, diazoxide-activated component of mitochondrial thallium uptake. Finally, the heart-derived cell line, H9C2, is protected from cell death stimuli by stable ROMK2 overexpression, whereas knockdown of the native ROMK exacerbates cell death. The findings support ROMK as the pore-forming subunit of the cytoprotective mitoK(ATP) channel.

Effects of mineralocorticoid and K+concentration on K+secretion and ROMK channel expression in a mouse cortical collecting duct cell line

The cortical collecting duct (CCD) plays a key role in regulated K(+) secretion, which is mediated mainly through renal outer medullary K(+) (ROMK) channels located in the apical membrane. However, the mechanisms of the regulation of urinary K(+) excretion with regard to K(+) balance are not well known. We took advantage of a recently established mouse CCD cell line (mCCD(cl1)) to investigate the regulation of K(+) secretion by mineralocorticoid and K(+) concentration. We show that this cell line expresses ROMK mRNA and a barium-sensitive K(+) conductance in its apical membrane. As this conductance is sensitive to tertiapin-Q, with an apparent affinity of 6 nM, and to intracellular acidification, it is probably mediated by ROMK. Overnight exposure to 100 nM aldosterone did not significantly change the K(+) conductance, while it increased the amiloride-sensitive Na(+) transport. Overnight exposure to a high K(+) (7 mM) concentration produced a small but significant increase in the apical membrane barium-sensitive K(+) conductance. The mRNA levels of all ROMK isoforms measured by qRT-PCR were not changed by altering the basolateral K(+) concentration but were decreased by 15-45% upon treatment with aldosterone (0.3 or 300 nM for 1 and 3 h). The paradoxical response of ROMK expression to aldosterone could possibly work as a preventative mechanism to avoid excessive K(+) loss which would otherwise result from the increased electrogenic Na(+) transport and associated depolarization of the apical membrane in the CCD. In conclusion, mCCD(cl1) cells demonstrate a significant K(+) secretion, probably mediated by ROMK, which is not stimulated by aldosterone but increased by overnight exposure to a high K(+) concentration.

The ROMK potassium channel is present in mammalian urinary tract epithelia and muscle

There is increasing evidence that mammalian urinary tract epithelial cells utilize membrane channels and transporters to transport solutes across their apical (luminal) and basalateral membranes to modify solute concentrations in both cell and urine. This study investigates the expression, localization, and regulation of the ROMK (K(ir) 1.1) potassium channels in rat and dog ureter and bladder tissues. Immunoblots of homogenates of whole ureter, whole bladder, bladder epithelial cells, and bladder smooth muscle tissues in both rat and dog identified approximately 45- to 50-kDa bands characteristic of ROMK in all tissues. RT-PCR identified ROMK mRNA in these same tissues in both animal species. ROMK protein localized by immunocytochemistry was strongly expressed in the apical membranes of the large umbrella cells lining the bladder lumen and to a lesser extent in the cytoplasm of epithelial cells and smooth muscle cells in the rat bladder. ROMK protein and mRNA were also discovered in cardiac, striated, and smooth muscle in diverse organs. There was no difference in immunoblot expression of ROMK abundance in bladder homogenates (whole bladder, epithelial cell, or muscle cell) or ureteral homogenates between groups of rats fed high- or low-potassium diets. Although the functional role of ROMK in urinary tract epithelia and smooth muscle is unknown, ROMK may participate in the regulation of epithelial and smooth muscle cell volume and osmolality, in the dissipation of potassium leaked or diffused from urine across the epithelial cell apical membranes or tight junctions, and in net or bidirectional potassium transport across urinary tract epithelia.

Regulation of ROMK (Kir 1.1) Channel Expression in Kidney Thick Ascending Limb by Hypertonicity: Role of TonEBP and MAPK Pathways

The present study assessed the mechanisms by which hypertonicity caused by NaCl enhances the renal outer medullary potassium channel (ROMK) mRNA abundance in rat kidney medullary thick ascending limb (MTAL) and in cultured mouse TAL cells. Using the run-off technique, we observed that the ROMK gene transcription rate in nuclei isolated from MTAL fragments was enhanced ∼40% by a high NaCl medium. In MTAL fragments, hypertonicity (450 mosm) caused by NaCl, not by mannitol or urea, enhanced both ROMK mRNA abundance and tonicity-responsive enhancer binding protein (TonEBP) total abundance and nuclear localization. In an immortalized mouse TAL cell culture in which ROMK is apically expressed, hypertonicity caused by both NaCl and mannitol, not urea, enhanced both ROMK mRNA abundance and TonEBP total abundance and nuclear localization. Confocal microscopy confirmed an increased nuclear translocation of TonEBP in response to NaCl-induced hypertonicity. Finally, inhibition of the p38 MAPK pathway by SB203580 and of the ERK pathway by PD98059 abolished the NaCl-induced stimulation of TonEBP and ROMK. These results establish that mRNA expression of ROMK is augmented in the MTAL by NaCl-induced hypertonicity through stimulation of ROMK gene transcription, and that TonEBP and the p38 MAPK and ERK pathways are involved in this effect.

Overexpression of regucalcin enhances its nuclear localization and suppressesL-type Ca2+ channel and calcium-sensing receptor mRNA expressions in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The effect of regucalcin (RC), a regulatory protein in intracellular signaling pathway, on the gene expression of various mineral ion transport-related proteins was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing RC. NRK52E cells (wild-type) and stable RC/pCXN2 transfectant were cultured for 72 h in medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured 24-72 h in a medium containing either vehicle, aldosterone (10(-8) or 10(-7) M), or parathyroid hormone (PTH) (1-34) (10(-8) or 10(-7) M) without BS. RC was markedly localized in the nucleus of transfectants. Overexpression of RC caused a significant increase in rat outer medullary K(+) channel (ROMK) mRNA expression, while it caused a remarkable decrease in L-type Ca(2+) channel and calcium-sensing receptor (CaR) mRNA expressions. Overexpression of RC did not have an effect on epithelial sodium channel (ENaC), Na, K-ATPase (alpha-subunit), Type II Na-Pi cotransporter (NaPi-IIa), angiotensinogen, Na(+)-Ca(2+) exchanger, and glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions. Hormonal effect on gene expression, moreover, was examined. Culture with aldosterone (10(-8) or 10(-7) M) caused a significant increase in ENaC, Na, K-ATPase, and ROMK mRNA expressions in the wild-type cells. Those increases were weakened in the transfectants. Culture with PTH (10(-8) or 10(-7) M) significantly decreased NaPi-IIa mRNA expression in the wild-type cells. This effect was not altered in the transfectants. PTH significantly decreased angiotensinogen mRNA expression in the wild-type cells and the transfectants, while aldosterone had no effect. Culture with PTH (10(-8) or 10(-7) M) caused a significant decrease in L-type Ca(2+) channel and CaR mRNA expressions in the wild-type cells, while the hormone significantly increased Na(+)-Ca(2+) exchanger mRNA expression. The effects of PTH on L-type Ca(2+) channel, CaR, and Na(+)-Ca(2+) exchanger mRNA expressions were also seen in the transfectants. This study demonstrates that overexpression of RC caused a remarkable increase in its nuclear localization, and that it has suppressive effects on the gene expression of L-type Ca(2+) channel or CaR, which regulates intracellular Ca(2+) signaling, among various regulator proteins for mineral ions in NRK52E cells.

Expression of the K+ channel Kir7.1 in the developing rat kidney: Role in K+ excretion

Coordinated expression of ROMK (luminal K+ channel in the thick ascending limb and the collecting duct) and Na+,K+-ATPase has been demonstrated to be involved in the postnatal development of renal K+ excretion; however, the developmental expression of the basolateral K+ channel Kir7.1 is unknown. The purpose of this study was to elucidate the possible involvement of Kir7.1 in the maturation of renal K+ excretion. Developmental changes in the renal K+ excretion under the condition of K+ overload was investigated by collecting urine from neonatal rats infused with K+ (KCl solution). RNase protection analysis was used to elucidate the expression of Kir7.1 and Na+,K+-ATPase mRNA in pre- and postnatal rats, and the expression of Kir7.1 and ROMK mRNA at 7, 14, and 21 days. Western blotting of Kir7.1, and immunohistochemistry of Kir7.1 and ROMK were used to determine their protein expression. The ratio of urinary K+ excretion to K+ load increased between 7 and 14 days after birth. In addition, half excretion time of K+ load gradually decreased through the experimental period of 7 and 21 days. Na+,K+-ATPase mRNA levels showed a peak of up-regulation at birth that remained elevated. ROMK1 mRNA levels significantly increased between 7 and 14 days. On the other hand, Kir7.1 mRNA and protein levels significantly increased between 14 and 21 days. Kir7.1 protein in the thick ascending limb was first recognized at 7 days, whereas its expression in the distal convoluted tubule and the cortical collecting duct was found in 21-day-old neonates. Our results suggest that Kir7.1 is involved in the development of renal K+ excretion between 14 and 21 days after birth under the condition of K+ overload.

Regulation by glucocorticoids and osmolality of expression of ROMK (Kir 1.1), the apical K channel of thick ascending limb.

Mechanisms of regulation of ROMK channel mRNA and protein expression in medullary thick ascending limb (MTAL) were assessed in rat MTAL fragments incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR and ROMK protein by immunoblotting analysis of crude membranes. Medium hyperosmolality (450 mosmol/kgH(2)O; NaCl plus urea added to isoosmotic medium) increased ROMK mRNA (P < 0.04) and protein (P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA (P < 0.02). Hyperosmolality and dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-d-arginine) vasopressin (1 nM) or 0.5 mM 8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However, 8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK mRNA in the isoosmotic but not in the hyperosmotic medium (P < 0.004). In in vivo studies, the abundance of ROMK protein and mRNA increased in adrenalectomized (ADX) rats infused with dexamethasone compared with ADX rats (P < 0.02). These results establish glucocorticoids and medium NaCl concentration as direct regulators of MTAL ROMK mRNA and protein expression, which may be modulated by cAMP-dependent factors.

Inhibition of protein-tyrosine phosphatase stimulates the dynamin-dependent endocytosis of ROMK1.

We have previously shown that inhibiting protein-tyrosine kinase increased whereas inhibiting protein-tyrosine phosphatase (PTP) decreased renal outer medullary potassium channel 1 (ROMK1) channel activity (1). We have now used confocal microscopy, the patch clamp technique, and biotin labeling to further examine the role of tyrosine phosphorylation in regulating ROMK1 trafficking. Human embryonic kidney 293 cells were cotransfected with c-Src and green fluorescent protein-ROMK1, which has the same biophysical properties as those of ROMK1. Patch clamp studies have shown that phenylarsine oxide (PAO), an inhibitor of PTP, decreased the activity of ROMK1. Moreover, addition of PAO reduced the cell surface localization of green fluorescent protein-ROMK1 detected by confocal microscopy and diminished the surface ROMK1 density by 65% measured by biotin labeling. Also, PAO treatment significantly increased the phosphorylation of ROMK1. The notion that the effect of PAO is mediated by stimulating tyrosine phosphorylation-induced endocytosis of ROMK1 has also been supported by findings that mutating the tyrosine residue 337 of ROMK1 to alanine abolished the effect of PAO. Finally, the inhibitory effect of PAO on ROMK1 was completely blocked in the cells co-transfected with dominant negative dynamin (dynaminK44A). This indicates that the tyrosine phosphorylation-induced endocytosis of ROMK1 is dynamin-dependent. We conclude that inhibiting PTP increases ROMK1 phosphorylation and results in a dynamin-dependent internalization of the channel.

Differential gene regulation of renal salt entry pathways by salt load in the distal nephron of the rat.

The aim of the present study was to determine the molecular responses of the main salt-reabsorbing systems in the distal nephron to changes of salt load of the organism. For this purpose we analysed messenger ribonucleic acid (mRNA) levels for the bumetanide-sensitive Na+K+2Cl- cotransporter (BSC1), the thiazide-sensitive Na+Cl- cotransporter (TSC), the kidney-specific inwards rectifier K+ channel (ROMK), the amiloride-sensitive epithelial Na+ channel (ENaC) and the kidney-specific Cl- channel ClC-K2, in the cortex and inner and outer medulla of kidneys from male Sprague-Dawley rats fed a high- (8% w/w), normal- (0.6%) or low-(0.02%) salt diet or treated chronically with subcutaneous infusions of furosemide (12 mg/kg per day). BSC1 and ROMK mRNA levels did not differ between the four treatment groups. TSC mRNA increased during furosemide treatment 1.75-fold versus control but was not affected by a high- or a low-salt diet. The mRNA for the alpha-subunit of ENaC increased with the low-salt diet (about 1.5-fold) and with furosemide (about 2.1-fold) in all kidney zones, but did not change with the high-salt diet. Dietary salt loading down-regulated CIC-K2 mRNA in the outer medulla 0.6-fold versus control whilst furosemide treatment, but not the low-salt diet, increased ClC-K2 mRNA in the outer (1.6-fold) and inner medulla (2.0-fold). These findings suggest that gene expression of Na+ and Cl- entry pathways in the distal nephron are at least partly regulated by the salt load of the organism, such that salt-reabsorbing systems are stimulated by salt deficiency and suppressed by salt overload.

Developmental expression of ROMK mRNA in rabbit cortical collecting duct.

The cortical collecting duct (CCD) is a major site of regulation of K+ homeostasis in the fully differentiated mammalian kidney. CCDs isolated from adult rabbits and microperfused in vitro secrete K+ into the tubular fluid at high rates. However, CCDs dissected from newborn animals show no significant net K+ secretion until the 3rd wk of life, at least in part because of a paucity of conducting apical secretory K+ (SK) channels. To determine whether the abundance of genes encoding the SK channel is developmentally regulated, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis to test for the presence of mRNA encoding rat outer medullary K+ channel (ROMK), considered to be a major subunit of the SK channel, in kidney and single CCDs isolated from maturing rabbits. Using rat ROMK-specific primers, RT-PCR of rabbit kidney yielded an amplification product of expected size and sequence. Northern blot analysis identified a single band at approximately 2.9 kb in kidney at all ages. Densitometric analysis revealed a progressive increase in steady state expression of ROMK message in kidney after birth. RT-PCR of individual CCDs yielded a single band of predicted size for ROMK in all segments isolated from animals > or =3 wk old. In contrast, transcripts were not detected in any CCD samples obtained from 1-wk-old animals and were identified in only 30% of CCD samples isolated from 2-wk-old rabbits. In all of the latter tubular samples, a specific PCR product of correct size for beta-actin mRNA was detected. These results suggest that an increase in steady state expression of ROMK mRNA contributes to the developmental appearance of conducting secretory K+ channels in the CCD.

Developmental expression of ROMK in rat kidney.

The apical secretory K+ (SK) channel in the principal cell represents the rate-limiting step for K+ secretion across the cortical collecting duct (CCD). Patch clamp analysis of maturing rabbit principal cells identifies an increase in number of conducting SK channels after the 2nd week of life [L. M. Satlin and L. G. Palmer. Am. J. Physiol. 272 (Renal Physiol. 41): F397-F404, 1997], approximately 1 wk after an increase in activity of the amiloride-sensitive epithelial Na+ channel (ENaC) is detected. To correlate the postnatal increase in channel activity with developmental expression of ROMK, the molecular correlate of the SK channel, we used gene-specific probes to show a developmental increase in abundance of renal ROMK mRNA and a ROMK-specific antibody to examine the nephron distribution, localization, and abundance of this protein in developing rat kidney. Using antibodies directed against aquaporin-3 (AQP-3) and Tamm-Horsfall protein (THP), we confirmed that ROMK was expressed along the apical membranes of principal cells and thick ascending limbs of Henle (TALH) in adult kidney. Within the midcortex of the neonatal kidney, ROMK-positive segments revealed weak coincident staining with the TALH-specific antibody but did not colabel with an antibody directed against distal and connecting tubule (CNT)-specific kallikrein or the lectin Dolichos biflorus agglutinin (DBA), which labels proximal tubules and collecting ducts. In inner cortex and outer medulla of kidneys from 1-wk-old animals, ROMK protein was identified in medullary TALH (MTALH) and DBA-positive collecting ducts. By 3 wk of age, coincident ROMK and DBA expression was detected in midcortical and outer cortical CNTs and CCDs. Immunoblot analysis of plasma membrane-enriched fractions of maturing rat kidney revealed a developmental increase in a approximately 40-kDa band, the expected size for ROMK. Immunolocalization of alpha-ENaC showed apical staining of a majority of cells in distal nephron segments after the 1st week of postnatal life. The beta- and gamma-ENaC subunit expression was routinely detected in a mostly cytoplasmic distribution immediately after birth, albeit in low abundance; gamma-ENaC showed some apical polarization. These results suggest that the postnatal increases in a principal cell apical SK and Na+ channel activity are mediated, at least in part, by increases in abundance of ROMK message and protein and ENaC subunit proteins.

Splicing of a retained intron within ROMK K+ channel RNA generates a novel set of isoforms in rat kidney.

The renal outer medulla K+ channel (ROMK) family of K+ channels may constitute a major pathway for K+ secretion in the distal nephron. To date, four main isoforms of this gene have been identified in the rat that differ only in their NH2-terminal amino acids and that share a common "core exon" that determines the remaining protein sequence. Using RT-PCR, we have identified a new set of ROMK isoforms in rat kidney that are generated by the deletion of a region within the ROMK core sequence that is identifiable as a typical mammalian intron. This splicing event was shown to be reproducible in vitro by detection of deleted ROMK mRNA in Madin-Darby canine kidney (MDCK) cells stably transfected with the gene for ROMK2. Translation of the deletion variant of ROMK2 was confirmed in vitro and visualized in MDCK cells following transient transfection with an enhanced green fluorescent protein tag. The deletion in this core region is predicted to generate hydrophilic proteins that are approximately one-third of the size of native ROMK and lack membrane-spanning domains.

Regulation of distal nephron K+ channels (ROMK) mRNA expression by aldosterone in rat kidney.

1. The expression of ROMK mRNA isoforms in rat kidney was measured using competitive polymerase chain reaction. Under basal conditions the expression of ROMK2 and 3 mRNA was significantly higher than that of ROMK1 or 6. 2. Administration of aldosterone for a period of 1 week significantly increased the mRNA expression not only of the alpha1-subunit of the Na+-K+-ATPase, but also of ROMK2, 3 and 6. 3. These data not only provide evidence that ROMK K+ channels may be involved with mineralocorticoid-sensitive K+ secretion in the distal nephron, but also demonstrate for the first time that ROMK6 may be involved in this process.

Developmental Expression of Na and K Channel Proteins in the Rat Renal Cortical Collecting Duct (CCD) † 1851

Growing organisms must retain Na and K. The final regulation of Na and K homeostasis occurs in the renal CCD where, in the adult, the amiloride-sensitive epithelial Na channel (ENaC) and low-conductance K-secretory (SK) channel constitute the rate-limiting steps for Na absorption and K secretion, respectively. Patch clamp analysis of principal cells in CCDs isolated from maturing rabbits identifies an increase in number of conducting apical Na channels after the 1st wk of postnatal life; a surge in SK channel activity, however, is not observed until after the 2nd wk of life. The purpose of the present study was to correlate the functional expression of channel activity with appearance of channel proteins in the developing kidney. To this end, we examined the nephron distribution and localization of ENaC and SK channels in cryosections of kidney using polyclonal antibodies raised against the α-, β-, and γ-subunits of rat ENaC (generously provided by P. Smith and C. Canessa) and the putative rat SK channel, ROMK (kindly provided by L. Palmer). Because the available antibodies did not recognize rabbit antigens, studies were performed in rat; Northern analyses revealed similar developmental patterns of expression of ROMK mRNA in rat and rabbit kidney. The identity of principal cells was confirmed by the selective binding of anti-aquaporin (AQP)-3 antibody; thick ascending limbs of Henle (TALHs) were identified with an anti-uromucoid antibody. Immuno-fluorescence studies using the anti-α ENaC antibody showed apical staining of a majority of cells in CCDs isolated from all animals >1 wk of age. Similarly, β- and γ-subunit expression was routinely detected, albeit in a predominantly cytoplasmic distribution, after the 1st wk of postnatal life. ROMK, absent in the mid-outer cortex of 1-wk-old animals, was observed in the inner cortex and medulla of these same animals, presumably in TALHs. By 3 wks of age, ROMK was easily detected along the apical membrane of a discrete population of TALH-negative cortical structures, believed to represent connecting and collecting tubules. Staining with immunoabsorbed serum or secondary antibody alone was negative. These results suggest that the postnatal surges in activity of principal cell Na and SK channels are mediated, in part, by increases in abundance of ENaC and ROMK proteins in the maturing CCD.

Dissociation of K channel density and ROMK mRNA in rat cortical collecting tubule during K adaptation.

The density of conducting K channels in the apical membrane of the rat cortical collecting tubule (CCT) is increased by a high-K diet. To see whether this involved increased abundance of mRNA coding for K channel protein, we measured the relative amounts of mRNA for ROMK, the clone of the gene thought to encode the secretory K channel in the CCT. Tubules were isolated and fixed for in situ hybridization with a probe based on the ROMK sequence. Radiolabeled probe associated with the tubule was quantified using densitometric analysis of the autoradiographic images of the tubules. The densitometry signal was shown to be proportional to the amount of radioactive probe in the sample and to the time of exposure of the film. The technique was able to detect an approximately twofold increase in the abundance of mRNA coding for the water channel aquaporin 3 (AQP3), in response to a 30-h dehydration period. Tubules from rats fed a normal diet or a high-K (10% KCl) diet had equal amounts of ROMK mRNA. This suggests that an increase in the abundance of mRNA does not underlie the increase in channel density observed under these conditions.

Localization of the ROMK protein on apical membranes of rat kidney nephron segments.

The ATP-sensitive, inwardly rectifying K+ channel, ROMK, has been suggested to be the low-conductance ATP-sensitive K+ channel identified in apical membranes of mammalian renal thick ascending limb (TAL) and cortical collecting duct (CCD). Mutations in the human ROMK gene (KIR 1.2) have been identified in kindreds with neonatal Bartter's syndrome. In the present study, we generated polyclonal antibodies raised against both a COOH-terminal (amino acids 252-391) ROMK-maltose binding protein (MBP) fusion protein and an NH2-terminal (amino acids 34-49) ROMK peptide. Affinity-purified anti-ROMK COOH-terminal antibody detected the 45-kDa ROMK protein in kidney tissues and HEK-293 cells transfected with ROMK1 cDNA. The antibody also recognized 85- to 90-kDa proteins in kidney tissue; these higher molecular weight proteins were abolished by immunoabsorption with ROMK-MBP fusion protein and were also detected on Western blots using anti-ROMK NH2-terminal antibody. Immunofluoresence studies using anti-ROMK COOH-terminal antibody showed intense apical staining along the loop of Henle and distal nephron; staining with preimmune and immunoabsorbed serum was negative. When colocalized with distal nephron markers [the thiazide-sensitive cotransporter (rTSC1), the bumetanide-sensitive cotransporter (rBSC1), the vacuolar type H(+)-ATPase, and neuronal nitric oxide synthase (NOS I)], the ROMK protein was found primarily at the apical border of cells in the TAL, macula densa, distal convoluted tubule, and connecting tubule. Within the CCD, the ROMK protein was expressed in principal cells and was absent from intercalated cells. The tubule localization and polarity of ROMK staining are consistent with the distribution of ROMK mRNA and provide more support for ROMK being the low-conductance K+ secretory channel in the rat distal nephron.

ROMK inwardly rectifying ATP-sensitive K+ channel. I. Expression in rat distal nephron segments

The inwardly rectifying, ATP-sensitive K+ channel (ROMK) was localized by in situ hybridization in the rat kidney. Tissue in situ hybridization revealed that transcripts encoding the ROMK channel were expressed predominantly in cortical and outer medullary nephron segments. The localization of ROMK mRNA to specific nephron segments was assessed by hybridization of isolated nephron segments with an ROMK-specific probe (single segment in situ hybridization). ROMK mRNA was present in cortical and medullary thick ascending limb, distal tubule, and cortical and outer medullary collecting ducts, but not in proximal tubule. A weak hybridization was observed with inner medullary collecting ducts. To confirm these results, serial cryosections were alternatively stained by hybridization histochemistry for ROMK mRNA or by immunocytochemistry using antibodies specific for S1, S2, or S3 proximal tubular segments. Tubular cells that displayed immunoreactivity with the proximal tubular segment-specific antibodies showed little, if any, ROMK message. In addition, using an in situ hybridization and immunocytochemistry double-labeling technique, ROMK transcripts and vitamin D-dependent calcium-binding protein were shown to colocalize to the distal tubule (distal convoluted tubule and connecting tubule). The overall nephron localization of ROMK mRNA shown in these studies is consistent with the possibility that this novel channel may represent the low-conductance ATP-sensitive K+ channel that has been identified in apical membranes of thick limb and collecting duct segments and is believed to participate in K+ secretion.