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

1995 ◽  
Vol 268 (6) ◽  
pp. F1124-F1131 ◽  
Author(s):  
W. S. Lee ◽  
S. C. Hebert
Keyword(s):  
In Situ Hybridization ◽  
Distal Tubule ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Double Labeling ◽  
Nephron Segments ◽  
Collecting Ducts ◽  
Proximal Tubular ◽  
Inwardly Rectifying

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.

Localization of rat CLC-K2 chloride channel mRNA in the kidney

1999 ◽  
Vol 276 (4) ◽  
pp. F552-F558 ◽  
Author(s):  
Momono Yoshikawa ◽  
Shinichi Uchida ◽  
Atsushi Yamauchi ◽  
Akiko Miyai ◽  
Yujiro Tanaka ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Chloride Channel ◽  
Rat Kidney ◽  
Water Channel ◽  
Physiological Role ◽  
Thick Ascending Limb ◽  
Nephron Segments ◽  
Henle's Loop ◽  
Collecting Ducts

To gain insight into the physiological role of a kidney-specific chloride channel, CLC-K2, the exact intrarenal localization was determined by in situ hybridization. In contrast to the inner medullary localization of CLC-K1, the signal of CLC-K2 in our in situ hybridization study was highly evident in the superficial cortex, moderate in the outer medulla, and absent in the inner medulla. To identify the nephron segments where CLC-K2 mRNA was expressed, we performed in situ hybridization of CLC-K2 and immunohistochemistry of marker proteins (Na+/Ca2+exchanger, Na+-Cl−cotransporter, aquaporin-2 water channel, and Tamm-Horsfall glycoprotein) in sequential sections of a rat kidney. Among the tubules of the superficial cortex, CLC-K2 mRNA was highly expressed in the distal convoluted tubules, connecting tubules, and cortical collecting ducts. The expression of CLC-K2 in the outer and inner medullary collecting ducts was almost absent. In contrast, a moderate signal of CLC-K2 mRNA was observed in the medullary thick ascending limb of Henle’s loop, but the signal in the cortical thick ascending limb of Henle’s loop was low. These results clearly demonstrated that CLC-K2 was not colocalized with CLC-K1 and that its localization along the nephron segments was relatively broad compared with that of CLC-K1.

Cloning and functional expression of human retinal Kir2.4, a pH-sensitive inwardly rectifying K+ channel

2000 ◽  
Vol 279 (3) ◽  
pp. C771-C784 ◽  
Author(s):  
Bret A. Hughes ◽  
Gyanendra Kumar ◽  
Yukun Yuan ◽  
Anuradha Swaminathan ◽  
Denise Yan ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Functional Expression ◽  
Neural Retina ◽  
Northern Analysis ◽  
K Channel ◽  
Metaphase Chromosomes ◽  
Preferential Expression ◽  
Kir Channel ◽  
Inwardly Rectifying

To identify novel potassium channel genes expressed in the retina, we screened a human retina cDNA library with an EST sequence showing partial homology to inwardly rectifying potassium (Kir) channel genes. The isolated cDNA yielded a 2,961-base pair sequence with the predicted open reading frame showing strong homology to the rat Kir2.4 (rKir2.4). Northern analysis of mRNA from human and bovine tissues showed preferential expression of Kir2.4 in the neural retina. In situ hybridization to sections of monkey retina detected Kir2.4 transcript in most retinal neurons. Somatic hybridization analysis and dual-color in situ hybridization to metaphase chromosomes mapped Kir2.4 to human chromosome 19 q13.1–q13.3. Expression of human Kir2.4 cRNA in Xenopus oocytes generated strong, inwardly rectifying K+ currents that were enhanced by extracellular alkalinization. We conclude that human Kir2.4 encodes an inwardly rectifying K+ channel that is preferentially expressed in the neural retina and that is sensitive to physiological changes in extracellular pH.

Expression of the mRNAs for the Kv3.1 potassium channel gene in the adult and developing rat brain

1992 ◽  
Vol 68 (3) ◽  
pp. 756-766 ◽  
Author(s):  
T. M. Perney ◽  
J. Marshall ◽  
K. A. Martin ◽  
S. Hockfield ◽  
L. K. Kaczmarek
Keyword(s):  
In Situ Hybridization ◽  
Rat Brain ◽  
K Channel ◽  
Rnase Protection ◽  
Hybridization Histochemistry ◽  
Adult Rat ◽  
In Situ Hybridization Histochemistry ◽  
Adult Rat Brain ◽  
Developing Rat

1. The gene for a mammalian Shaw K+ channel has recently been cloned and has been shown, by alternative splicing, to give rise to two different transcripts, Kv3.1 alpha and Kv3.1 beta. To determine whether these channels are associated with specific types of neurons and to determine whether or not the alternately spliced K+ channel variants are differentially expressed, we used ribonuclease (RNase) protection assays and in situ hybridization histochemistry to localize the specific subsets of neurons containing Kv3.1 alpha and Kv3.1 beta mRNAs in the adult and developing rat brain. 2. In situ hybridization histochemistry revealed a heterogeneous expression pattern of Kv3.1 alpha mRNA in the adult rat brain. Highest Kv3.1 alpha mRNA levels were expressed in the cerebellum. High levels of hybridization were also detected in the globus pallidus, subthalamus, and substantia nigra reticulata. Many thalamic nuclei, but in particular the reticular thalamic nucleus, hybridized well to Kv3.1 alpha-specific probes. A subpopulation of cells in the cortex and hippocampus, which by their distribution and number may represent interneurons, were also found to contain high levels of Kv3.1 alpha mRNA. In the brain stem, many nuclei, including the inferior colliculus and the cochlear and vestibular nuclei, also express Kv3.1 alpha mRNA. Low or undetectable levels of Kv3.1 alpha mRNA were found in the caudate-putamen, olfactory tubercle, amygdala, and hypothalamus. 3. Kv3.1 beta mRNA was also detected in the adult rat brain by both RNase protection assays and by in situ hybridization experiments. Although the beta splice variant is expressed at lower levels than the alpha species, the overall expression pattern for both mRNAs is similar, indicating that both splice variants co-expressed in the same neurons. 4. The expression of Kv3.1 alpha and Kv3.1 beta transcripts was examined throughout development. Kv3.1 alpha mRNA is detected as early as embryonic day 17 and then increases gradually until approximately postnatal day 10, when there is a large increase in the amount of Kv3.1 alpha mRNA. Interestingly, the expression of Kv3.1 beta mRNA only increases gradually during the developmental time frame examined. Densitometric measurements indicated that Kv3.1 alpha is the predominant splice variant found in neurons of the adult brain, whereas Kv3.1 beta appears to be the predominant species in embryonic and perinatal neurons. 5. Most of the neurons that express the Kv3.1 transcripts have been characterized electrophysiologically to have narrow action potentials and display high-frequency firing rates with little or no spike adaptation.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney

2010 ◽  
Vol 299 (6) ◽  
pp. F1496-F1506 ◽  
Author(s):  
Alan C. Pao ◽  
Aditi Bhargava ◽  
Francesca Di Sole ◽  
Raymond Quigley ◽  
Xinli Shao ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Proximal Tubule ◽  
Cell Surface Expression ◽  
Thick Ascending Limb ◽  
Proximal Tubule Cells ◽  
Mammalian Kidney ◽  
Tubule Cells ◽  
Exchange Activity

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na+) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na+/H+ exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na+/H+ exchange activity by Na+-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na+/H+ exchange activity by >30%. Moreover, the sgk2-mediated increase in Na+/H+ exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na+ transport through NHE3 in the proximal tubule.

scholarly journals Differential Expression Patterns of Claudins, Tight Junction Membrane Proteins, in Mouse Nephron Segments

2002 ◽  
Vol 13 (4) ◽  
pp. 875-886 ◽  
Author(s):  
Yumiko Kiuchi-Saishin ◽  
Shimpei Gotoh ◽  
Mikio Furuse ◽  
Akiko Takasuga ◽  
Yasuo Tano ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Tight Junction ◽  
Polyclonal Antibodies ◽  
Collecting Duct ◽  
Expression Patterns ◽  
Distal Tubule ◽  
Northern Blotting ◽  
Thick Ascending Limb ◽  
Specific Expression ◽  
Nephron Segments

ABSTRACT. As the first step in understanding the physiologic functions of claudins (tight junction integral membrane proteins) in nephrons, the expression of claudin-1 to -16 in mouse kidneys was examined by Northern blotting. Among these claudins, only claudin-6, -9, -13, and -14 were not detectable. Claudin-5 and -15 were detected only in endothelial cells. Polyclonal antibodies specific for claudin-7 and -12 were not available. Therefore, the distributions of claudin-1, -2, -3, -4, -8, -10, -11, and -16 in nephron segments were examined with immunofluorescence microscopy. For identification of individual segments, antibodies specific for segment markers were used. Immunofluorescence microscopic analyses of serial frozen sections of mouse kidneys with polyclonal antibodies for claudins and segment markers revealed that claudins demonstrated very complicated, segment-specific, expression patterns in nephrons, i.e., claudin-1 and -2 in Bowman’s capsule, claudin-2, -10, and -11 in the proximal tubule, claudin-2 in the thin descending limb of Henle, claudin-3, -4, and -8 in the thin ascending limb of Henle, claudin-3, -10, -11, and -16 in the thick ascending limb of Henle, claudin-3 and -8 in the distal tubule, and claudin-3, -4, and -8 in the collecting duct. These segment-specific expression patterns of claudins are discussed, with special reference to the physiologic functions of tight junctions in nephrons.

Intracellular localization of types I and II collagen mRNA and endoplasmic reticulum in embryonic corneal epithelia

1991 ◽  
Vol 100 (1) ◽  
pp. 23-33 ◽  
Author(s):  
K.K. Svoboda
Keyword(s):  
Endoplasmic Reticulum ◽  
In Situ Hybridization ◽  
Laser Scanning ◽  
Intracellular Localization ◽  
Confocal Laser ◽  
Type I ◽  
Basal Cells ◽  
Double Labeling ◽  
Collagen Mrna

The intracellular distribution of endoplasmic reticulum (ER) and types I and II collagen mRNA was analyzed in whole-mount preparations of freshly isolated corneal epithelia using in situ hybridization combined with confocal laser scanning analysis. The ER stained with DiOC6 (3) was prominent in both the periderm and basal cells. The basal cell ER distribution was perinuclear in the center of the cells, but below the nucleus the ER occupied nearly all of the cytoplasm in a reticular pattern similar to that seen with TEM cross-sections. Initial single label in situ hybridization studies showed that both the periderm and basal cells were positive for both types I and II collagen mRNA. The collagen cDNA probes appeared perinuclear in the center of the basal cells, similar to the DiOC6(3) staining pattern. In double-labeling experiments, the two mRNAs that translate chains of type I collagen, alpha 1 and alpha 2, colocalized within the same cell. However, the hybridization of probes specific for type I and II collagen mRNAs had separate, but overlapping, distributions within the same cell.

scholarly journals Expression of the c-fos protooncogene by human and murine erythroblasts

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 947-951 ◽  
Author(s):  
JF Caubet ◽  
MT Mitjavila ◽  
A Dubart ◽  
D Roten ◽  
SC Weil ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Northern Blotting ◽  
Hematopoietic Tissue ◽  
Double Labeling ◽  
Murine Bone Marrow ◽  
Marrow Cells

Abstract The expression of the c-fos protooncogene was investigated by in situ hybridization in normal murine bone marrow cells. A strong signal was found in murine marrow cells having the morphologic features of erythroblasts. This result was confirmed in human marrow cells using a double labeling technique (in situ hybridization and immunocytochemistry). A majority (70%) of the cells expressing c-fos mRNA were glycophorin A-positive. In contrast, granulocytic precursors (CD 15-positive) or monocytes and their precursors (CD 14-positive cells) did not significantly hybridize with the c-fos probe. In addition, c-fos mRNA (2.2Kb) was detected by Northern blotting in RNA extracted from homogeneous populations of erythroblasts obtained by immune panning from fetal liver and from adult blood BFU-E-derived colonies. Fos protein was also detected in erythroblasts by immunofluorescence. The high level of c-fos mRNA previously found in hematopoietic tissue should therefore be related to the transcription of the c-fos gene during terminal erythroid differentiation.

scholarly journals Autoradiographic visualization of 35S-labeled cRNA probes combined with immunoperoxidase detection of choleragenoid: a double-labeling light microscopic method for in situ hybridization and retrograde tract tracing.

1994 ◽  
Vol 42 (6) ◽  
pp. 827-831 ◽  
Author(s):  
O Hermanson ◽  
H Ericson ◽  
G Sanchez-Watts ◽  
A G Watts ◽  
A Blomqvist
Keyword(s):  
In Situ Hybridization ◽  
Cobalt Acetate ◽  
Reaction Products ◽  
Double Labeling ◽  
Cholera Toxin Subunit B ◽  
Efferent Projections ◽  
Labeled Rna ◽  
Subunit B ◽  
Silver Grains

We describe a protocol for simultaneous light microscopic visualization of a neuron's efferent projections and its expression of mRNA. We have combined immunohistochemical visualization of the retrograde marker cholera toxin subunit B (CTb) with autoradiographic visualization of 35S-labeled cRNA probes. Injections of CTb were made into rat brain. Immunoreactivity for CTb was demonstrated by modification of the peroxidase-anti-peroxidase immunohistochemical technique, with DAB and nickel ammonium sulfate or cobalt acetate as chromogen. On the same sections, in situ hybridization was performed with a 35S-labeled RNA probe complementary to preproenkephalin mRNA or tyrosine hydroxylase mRNA. Many double-labeled neurons were detected. These neurons contained peroxidase reaction product and were covered by an accumulation of silver grains in the overlaying emulsion layer. The present method has several advantages over double-labeling methods using the combination of fluorescent tracers and oligonucleotide probes. Both reaction products are permanent and can be visualized simultaneously by light microscopy. Furthermore, both CTb and cRNA probes are very sensitive markers. In addition, the sections can be counterstained.

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