Protein phosphatase-1 in the kidney: evidence for a role in the regulation of medullary Na(+)-K(+)-ATPase

1995 ◽  
Vol 269 (5) ◽  
pp. F673-F680 ◽  
Author(s):  
D. Li ◽  
A. Aperia ◽  
G. Celsi ◽  
E. F. da Cruz e Silva ◽  
P. Greengard ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Hormonal Regulation ◽  
Transitional Epithelium ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Outer Cortex ◽  
Outer Medulla ◽  
Endogenous Dopamine ◽  
Outer Stripe

Previous studies of hormonal regulation of renal Na(+)-K(+)-ATPase have indicated that the activity of the sodium pump is regulated by phosphorylation-dephosphorylation reactions. Here we report that okadaic acid (OA) and calyculin A (CL-A), inhibitors of protein phosphatase (PP)-1 and PP-2A, inhibited Na(+)-K(+)-ATPase activity in cells from the rat thick ascending limb (TAL) of loop of Henle in a dose-dependent manner. CL-A was 10-fold more potent than OA. On the basis of the inhibitory constant values of CL-A and OA for PP-1 and PP-2A, it is concluded that the tubular effect is mainly due to inhibition of PP-1. In situ hybridization studies with oligonucleotide probes revealed very strong PP-1 alpha and PP-1 gamma 1 mRNA labeling in the outer stripe of the outer medulla, strong labeling in the inner stripe of the outer medulla, and weak labeling in the inner medulla. Very weak labeling was demonstrated in the outer cortex. PP-1 beta mRNA labeling was very strong in the inner stripe of the outer medulla, whereas the outer stripe had weaker labeling, and the inner medulla had weak labeling. PP-1 alpha, PP-1 beta, and PP-1 gamma 1 mRNA were also demonstrated in the transitional epithelium of the ureter. The abundance of the PP-1 alpha and PP-1 gamma isoforms as measured by immunoblotting was very high in tissue from the outer medulla, which also has a high abundance of the endogenous dopamine-regulated PP-1 inhibitor, DARPP-32.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter

1995 ◽  
Vol 269 (3) ◽  
pp. F405-F418 ◽  
Author(s):  
P. Igarashi ◽  
G. B. Vanden Heuvel ◽  
J. A. Payne ◽  
B. Forbush
Keyword(s):  
In Situ Hybridization ◽  
Alternative Splicing ◽  
Protein Kinase ◽  
Thick Ascending Limb ◽  
Outer Medulla ◽  
Donor And Acceptor ◽  
Outer Stripe ◽  
Dependent Protein ◽  
Isoform B

A full-length cDNA encoding the murine renal Na-K-Cl cotransporter (NKCC2) was cloned using library screening and anchored polymerase chain reaction. The deduced protein sequence contained 1,095 amino acids and was 93.5% identical to rabbit NKCC2 and 97.6% identical to rat BSC1. Two potential sites of phosphorylation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase and seven potential sites of phosphorylation by protein kinase C, which were previously identified in the rabbit and rat sequences, were phylogenetically conserved in the mouse. The expression of NKCC2 in the mouse was examined with Northern blot analysis and in situ hybridization. Expression of NKCC2 was kidney specific in both adult and embryonic mice. In the developing metanephros, NKCC2 was induced at 14.5 days post coitus and was expressed in distal limbs of immature loops of Henle but was absent from the ureteric bud, S-shaped bodies, and earlier nephrogenic structures. Similar to the rabbit, isoforms of NKCC2 that differed in the sequence of a 96-bp segment were identified in the mouse. In situ hybridization revealed that the isoforms exhibited different patterns of expression in the mature thick ascending limb of the loop of Henle as follows: isoform F was most highly expressed in the inner stripe of outer medulla, isoform A was most highly expressed in the outer stripe of the outer medulla, and isoform B was most highly expressed in the cortical thick ascending limb. To verify that the isoforms were generated by alternative splicing of mutually exclusive cassette exons, genomic clones encoding murine NKCC2 were characterized. Cassette exons were identified that corresponded to each of the three isoforms and were flanked by consensus splice donor and acceptor sequences.

Distribution and oligomeric association of splice forms of Na+-K+-ATPase regulatory γ-subunit in rat kidney

2002 ◽  
Vol 282 (3) ◽  
pp. F393-F407 ◽  
Author(s):  
Elena Arystarkhova ◽  
Randall K. Wetzel ◽  
Kathleen J. Sweadner
Keyword(s):  
Splice Variants ◽  
Rat Kidney ◽  
Thick Ascending Limb ◽  
Outer Medulla ◽  
Α Subunit ◽  
Proximal Tubule Cells ◽  
Γ Subunit ◽  
Outer Stripe ◽  
A Minor ◽  
Splice Forms

Renal Na+-K+-ATPase is associated with the γ-subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, γa and γb. Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the α-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na+-K+-ATPase conformation. In the cortex, the proportion of the γb-subunit was markedly lower, and the γa-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the γb-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na+-K+-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the γb-subunit. Because different mechanisms maintain and regulate Na+ homeostasis in different nephron segments, the splice forms of the γ-subunit may have evolved to control the renal Na+ pump through pump properties, gene expression, or both.

Immunolocalization of electroneutral Na-HCO3 − cotransporter in rat kidney

2000 ◽  
Vol 279 (5) ◽  
pp. F901-F909 ◽  
Author(s):  
Henrik Vorum ◽  
Tae-Hwan Kwon ◽  
Christiaan Fulton ◽  
Brian Simonsen ◽  
Inyeong Choi ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Electron Microscopic Level ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Electron Microscopic ◽  
Outer Medulla ◽  
Outer Stripe ◽  
Medullary Collecting Duct

An electroneutral Na-HCO3 − cotransporter (NBCN1) was recently cloned, and Northern blot analyses indicated its expression in rat kidney. In this study, we determined the cellular and subcellular localization of NBCN1 in the rat kidney at the light and electron microscopic level. A peptide-derived antibody was raised against the COOH-terminal amino acids of NBCN1. The affinity-purified antibody specifically recognized one band, ∼180 kDa, in rat kidney membranes. Peptide- N-glycosidase F deglycosylation reduced the band to ∼140 kDa. Immunoblotting of membrane fractions from different kidney regions demonstrated strong signals in the inner stripe of the outer medulla (ISOM), weaker signals in the outer stripe of the outer medulla and inner medulla, and no labeling in cortex. Immunocytochemistry demonstrated that NBCN1 immunolabeling was exclusively observed in the basolateral domains of thick ascending limb (TAL) cells in the outer medulla (strongest in ISOM) but not in the cortex. In addition, collecting duct intercalated cells in the ISOM and in the inner medulla also exhibited NBCN1 immunolabeling. Immunoelectron microscopy demonstrated that NBCN1 labeling was confined to the basolateral plasma membranes of TAL and collecting duct type A intercalated cells. Immunolabeling controls were negative. By using 2,7-bis-carboxyethyl-5,6-caboxyfluorescein, intracellular pH transients were measured in kidney slices from ISOM and from mid-inner medulla. The results revealed DIDS-sensitive, Na- and HCO3 −-dependent net acid extrusion only in the ISOM but not in mid-inner medulla, which is consistent with the immunolocalization of NBCN1. The localization of NBCN1 in medullary TAL cells and medullary collecting duct intercalated cells suggests that NBCN1 may be important for electroneutral basolateral HCO3 − transport in these cells.

Enhanced transcription of metallothionein genes in rat kidney: effect of uninephrectomy and compensatory renal growth

1995 ◽  
Vol 268 (4) ◽  
pp. F643-F650 ◽  
Author(s):  
R. K. Zalups ◽  
J. Fraser ◽  
J. Koropatnick
Keyword(s):  
Renal Cortex ◽  
Rat Kidney ◽  
Hepatic Tissue ◽  
Renal Growth ◽  
Outer Medulla ◽  
Compensatory Renal Growth ◽  
Intracellular Regulation ◽  
Outer Stripe ◽  
Remnant Kidney

Metallothioneins (MTs) have been implicated in the intracellular regulation of essential metals in eukaryotic cells, and increased expression of MT genes has been demonstrated during the growth and proliferation of cells. To explore the expression of MT in somatic cells undergoing growth (hypertrophy) in the kidney in situ, we measured the rates of transcription of the genes for MT-1 and MT-2, measured the levels of mRNA for MT-1 and MT-2, and measured the concentration of MT-1 and MT-2 protein in samples of renal (and hepatic) tissue from uninephrectomized (NPX) and sham-operated (SO) rats 15 days after surgery. The rates of transcription of the genes for MT-1 and MT-2 were found to be enhanced significantly in the remnant renal mass, particularly in the cortex and outer stripe of the outer medulla, and in the liver, after uninephrectomy and after 15 days allowing for compensatory renal growth. Increased accumulation of mRNA for MT-1 and MT-2 also occurred in the cortex and outer stripe of the outer medulla of the remnant kidney and in the liver in the NPX rats. Increased concentration of MT-1 and MT-2 protein (measured by radioimmunoassay), at the level of the whole kidney, renal cortex, and liver, was another feature detected in rats after uninephrectomy and 15 days of compensatory renal growth. These findings indicate that compensatory renal growth in response to uninephrectomy is associated with the induction of the expression of MT genes in the renal cortex and outer stripe of the outer medulla, as well as in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-specific regulation of cytosolic aspartate aminotransferase by glucocorticoids in the rat kidney

1993 ◽  
Vol 265 (5) ◽  
pp. C1298-C1305 ◽  
Author(s):  
S. Feilleux-Duche ◽  
M. Garlatti ◽  
M. Aggerbeck ◽  
M. Poyard ◽  
J. Bouguet ◽  
...  
Keyword(s):  
Aspartate Aminotransferase ◽  
Hormonal Regulation ◽  
Renal Cortex ◽  
Rat Kidney ◽  
Thick Ascending Limb ◽  
Outer Medulla ◽  
Maximal Increase ◽  
Basal Expression ◽  
Specific Increase ◽  
Specific Regulation

The basal expression and hormonal regulation of cytosolic aspartate aminotransferase (cAspAT) were investigated in the rat kidney. In adrenalectomized animals, the basal activity was highest in the renal cortex and in the inner stripe of the outer medulla (0.1-0.15 U/mg protein). The glucocorticoid analogue dexamethasone increased cAspAT activity about twofold in the cortex and in the inner stripe of the outer medulla but not in the papilla. A half-maximal increase in the activity was achieved at doses of approximately 5 micrograms/100 g body wt. The mineralocorticoid aldosterone did not modify the cAspAT activity. The cell specificity of the hormonal regulation was analyzed by in situ hybridization. In untreated adrenalectomized rats, a cAspAT cRNA probe labeled mainly the inner stripe of the outer medulla. After dexamethasone or hydrocortisone treatment, labeling was uniformly increased in this part of the medulla and was heterogeneously increased in the renal cortex. The specific increase in labeling within the cortex was shown to be confined to the distal convoluted tubule and the thick ascending limb. We conclude that, in addition to widespread basal expression, cAspAT is regulated by glucocorticoids in a highly cell-specific manner in the renal cortex. The enzyme may thus participate in the increased energy metabolism elicited by these hormones in these cells.

Renal expression of novel Na+/H+exchanger isoform NHE8

2003 ◽  
Vol 284 (3) ◽  
pp. F467-F473 ◽  
Author(s):  
Sunita Goyal ◽  
Gregory Vanden Heuvel ◽  
Peter S. Aronson
Keyword(s):  
Proximal Tubule ◽  
Amino Acid Identity ◽  
Outer Medulla ◽  
Reading Frame ◽  
Mouse Tissues ◽  
Outer Stripe ◽  
Apical Membranes ◽  
Nhe Isoforms ◽  
Renal Expression

Although Na+/H+exchanger isoform 3 (NHE3) mediates most Na+/H+exchange in the proximal tubule, studies of NHE3/NHE2 null mice suggest residual Na+-dependent proton secretion (Choi JY, Shah M, Lee MG, Schultheis PJ, Shull GE, Muallem S, and Baum M. J Clin Invest 105: 1141–1146, 2000). To characterize additional NHE isoforms that might be expressed in the kidney, we identified the partial sequence of a novel NHE. PCR was used to define the 5′- and 3′-ends, and a cDNA encoding the complete open reading frame was amplified from mouse kidney. The predicted protein of 576 amino acids, which we have named NHE8, has 30–35% amino acid identity to known mammalian isoforms (NHE1–7) but has >50% identity to Drosophila melanogaster “NHE1,” suggesting it is the mammalian ortholog of this ancient invertebrate isoform. Northern blot of mouse tissues revealed ubiquitous expression. Western blot using anti-NHE8 antibodies demonstrated protein expression in apical membranes purified from rat renal cortex by divalent cation precipitation. In situ hybridization revealed that NHE8 message was present in both cortex and medulla. In the cortex, NHE8 was present in the majority of cortical tubules, consistent with proximal tubule (S1 and S2) localization. In the medulla, NHE8 message was most highly expressed in the proximal tubules (S3) of the outer stripe of the outer medulla. Thus NHE8 is expressed in the proximal tubule, where it may contribute to apical membrane ion transport.

scholarly journals Differential localization patterns of Claudin 10, 16 and 19 in human, mouse, and rat renal tubular epithelia

2021 ◽  
Author(s):  
Caroline Prot-Bertoye ◽  
Camille Griveau ◽  
Karsten Skjødt ◽  
Lydie Cheval ◽  
Gaëlle Brideau ◽  
...  
Keyword(s):  
Tight Junction ◽  
Genetic Variations ◽  
Thick Ascending Limb ◽  
Loss Of Function ◽  
Outer Medulla ◽  
Outer Stripe ◽  
Proximal Tubular ◽  
Familial Hypomagnesemia ◽  
Weak Expression ◽  
Renal Tubular

Functional properties of the paracellular pathway depend critically on the set of claudins expressed at the tight junction. Two syndromes are causally linked to loss-of-function mutations of claudins: HELIX syndrome caused by genetic variations in the CLDN10 gene, and Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis caused by genetic variations in the CLDN16 or the CLDN19 gene. All three genes are expressed in the kidney, particularly in the thick ascending limb (TAL). However, localization of these claudins in humans and rodents remains to be delineated in detail. We studied the segmental and subcellular expression of CLDN10, 16 and 19 in both paraffin-embedded and frozen kidney sections from adult human, mouse and rat, using immunohistochemistry and immunofluorescence, respectively. Here CLDN10 was present in a subset of medullary and cortical TAL cells, localizing to basolateral domains and tight junction in human and rodent kidney. A weak expression was detected at the tight junction of proximal tubular cells. CLDN16 was primarily expressed in a subset of TAL cells in cortex and outer stripe of outer medulla, restricted to basolateral domains and tight junctional structures in both human and rodent kidney. CLDN19 predominantly colocalized with CLDN16 in tight junctions and basolateral domains of TAL but was also found in basolateral and junctional domains in more distal sites. CLDN10 expression at tight junction almost never overlapped with that of CLND16 and CLDN19, consistent with distinct junctional pathways with different permeation profiles in both human and rodent kidney.

Multihormonal regulation of nephron epithelia: achieved through combinational mode?

1995 ◽  
Vol 269 (4) ◽  
pp. R739-R748 ◽  
Author(s):  
C. De Rouffignac
Keyword(s):  
Sodium Chloride ◽  
Hormonal Regulation ◽  
Biological Effects ◽  
Basolateral Membrane ◽  
Positive Interactions ◽  
Thick Ascending Limb ◽  
Transport Pathways ◽  
Receptor Complexes ◽  
Nephron Segment ◽  
Urinary Concentrating Ability

The kidney is the main organ regulating composition of body fluids. A considerable number of hormones control the activity of renal cells to maintain hydromineral equilibrium. It becomes more and more difficult to interpret this multihormonal control in terms of regulatory processes. To illustrate this complexity, the hormonal regulation of electrolyte transport in the nephron thick ascending limb is taken as an example. This nephron segment is largely responsible for two kidney functions: the urinary-concentrating ability (by its capacity to deliver hypertonic sodium chloride into the medullary interstitium) and regulation of magnesium excretion into final urine. Six hormones are presently identified as acting on the transport of both sodium chloride and magnesium ions in this nephron segment. Therefore, the pertinent question is how the thick ascending limb and, hence, the kidney, is capable of regulating water balance independently from magnesium balance. It is proposed that the hormones act in combination: circulating levels of the individual hormones acting on these cells may determine the configuration of the paracellular and transcellular transport pathways of the epithelium either in the “sodium” or “magnesium” mode. The configuration would depend on the distribution and activity of the receptor at the surface of the basolateral membrane in contact with the circulating hormones. This distribution along with stimulation of respective signal transduction pathways would lead to the final biological effects. It is already known that the distribution of cell receptors may vary according to factors such as age, nutritional variability, hormonal status, degree of desensitization of the receptors, etc. The modulation of hormonal responses would depend therefore on the degree of coupling of hormone-receptor complexes to different intracellular transduction pathways and on the resulting negative and/or positive interactions between these pathways.

scholarly journals Exposure to Electromagnetic Fields (EMF) from Submarine Power Cables Can Trigger Strength-Dependent Behavioural and Physiological Responses in Edible Crab, Cancer pagurus (L.)

2021 ◽  
Vol 9 (7) ◽  
pp. 776
Author(s):  
Kevin Scott ◽  
Petra Harsanyi ◽  
Blair A. A. Easton ◽  
Althea J. R. Piper ◽  
Corentine M. V. Rochas ◽  
...  
Keyword(s):  
Electromagnetic Field ◽  
Policy Making ◽  
Physiological Responses ◽  
Dependent Manner ◽  
Power Cables ◽  
Environmental Assessments ◽  
Cancer Pagurus ◽  
Commercially Important ◽  
Total Haemocyte Count

The current study investigated the effects of different strength Electromagnetic Field (EMF) exposure (250 µT, 500 µT, 1000 µT) on the commercially important decapod, edible crab (Cancer pagurus, Linnaeus, 1758). Stress related parameters were measured (l-Lactate, d-Glucose, Total Haemocyte Count (THC)) in addition to behavioural and response parameters (shelter preference and time spent resting/roaming) over 24 h periods. EMF strengths of 250 µT were found to have limited physiological and behavioural impacts. Exposure to 500 µT and 1000 µT were found to disrupt the l-Lactate and d-Glucose circadian rhythm and alter THC. Crabs showed a clear attraction to EMF exposed (500 µT and 1000 µT) shelters with a significant reduction in time spent roaming. Consequently, EMF emitted from MREDs will likely affect crabs in a strength-dependent manner thus highlighting the need for reliable in-situ measurements. This information is essential for policy making, environmental assessments, and in understanding the impacts of increased anthropogenic EMF on marine organisms.

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