Phosphate-dependent glutaminase activity in rat renal cortical and medullary tubule segments

1990 ◽  
Vol 259 (6) ◽  
pp. F961-F970 ◽  
Author(s):  
P. A. Wright ◽  
M. A. Knepper
Keyword(s):  
Collecting Duct ◽  
Proximal Tubules ◽  
Thick Ascending Limb ◽  
Loop Of Henle ◽  
Ammonium Accumulation ◽  
Ammonium Production ◽  
Medullary Collecting Duct ◽  
Acid Loading ◽  
Glutaminase Activity ◽  
Convoluted Tubules

To determine whether local production of ammonium by medullary renal tubule segments may contribute to medullary ammonium accumulation, we measured activities of phosphate-dependent glutaminase (PDG) in microdissected tubule segments from rat medulla and cortex. PDG activities were very low in medullary loop of Henle segments but surprisingly high in inner medullary collecting duct (IMCD). In cortex, PDG levels were highest in distal convoluted tubule and cortical thick ascending limb, but substantial levels were also found in proximal segments, as reported previously. To determine effects of acid loading and alkali loading on PDG activity, 0.28 M NH4Cl (acid) or 0.28 M NaHCO3 (alkali) was added to rats' drinking water for 7 days. PDG activities in medullary segments were not affected by acid or alkali intake. Acid intake by rats increased PDG activity in S1 and S2 proximal convoluted tubules severalfold but did not affect the other cortical segments. We conclude that medullary loop of Henle segments probably contribute relatively little to medullary ammonium accumulation because of their low activities. The high PDG activity in IMCD suggests that ammonium could be produced and secreted by this segment. However, because total tubule length of IMCD is very low compared with proximal tubules, it appears unlikely that IMCD contributes substantially to overall renal ammonium production. PDG activity is regulated only in S1 and S2 proximal tubules, consistent with the view that the proximal tubule is the major site of regulation of renal ammonium production.

Lactate production in isolated segments of the rat nephron

1985 ◽  
Vol 248 (4) ◽  
pp. F522-F526 ◽  
Author(s):  
S. Bagnasco ◽  
D. Good ◽  
R. Balaban ◽  
M. Burg
Keyword(s):  
Collecting Duct ◽  
Lactate Production ◽  
Proximal Tubules ◽  
Antimycin A ◽  
Anaerobic Glycolysis ◽  
Metabolic Substrate ◽  
Collecting Ducts ◽  
Medullary Collecting Duct ◽  
Convoluted Tubules

Lactate production was measured directly in individual segments of the rat nephron. Tubules were dissected and then incubated in vitro with glucose as the only metabolic substrate. Each segment was incubated with and without antimycin A, an inhibitor of oxidative metabolism. Proximal tubules produced no lactate with or without antimycin A. The distal segments all produced lactate. The rate of lactate production without antimycin A ranged from 0.4 to 0.9 pmol X min-1 X mm-1 in all distal segments except one, the inner medullary collecting duct, which produced lactate at the significantly higher rate of 2.8 pmol X min-1 X mm-1. Antimycin A increased lactate production significantly in all of the distal segments. The increase was largest in medullary thick ascending limbs (1,400%) and cortical (798%) and outer medullary collecting ducts (357%). Increments were smaller in cortical thick ascending limbs (98%) and distal convoluted tubules (98%) and least in the inner medullary collecting ducts (28%). We conclude that lactate production occurs only in distal segments of the nephron and that under anoxic conditions significant amounts of ATP are produced by anaerobic glycolysis in these segments.

Long-term regulation of renal Na-dependent cotransporters and ENaC: response to altered acid-base intake

2000 ◽  
Vol 279 (3) ◽  
pp. F459-F467 ◽  
Author(s):  
Gheun-Ho Kim ◽  
Stephen W. Martin ◽  
Patricia Fernández-Llama ◽  
Shyama Masilamani ◽  
Randall K. Packer ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Na Channel ◽  
Thick Ascending Limb ◽  
Acid Base Balance ◽  
Acid Base ◽  
Opposite Pattern ◽  
Α Subunit ◽  
Base Balance ◽  
Acid Loading

Increased systemic acid intake is associated with an increase in apical Na/H exchange in the renal proximal tubule mediated by the type 3 Na/H exchanger (NHE3). Because NHE3 mediates both proton secretion and Na absorption, increased NHE3 activity could inappropriately perturb Na balance unless there are compensatory changes in Na handling. In this study, we use semiquantitative immunoblotting of rat kidneys to investigate whether acid loading is associated with compensatory decreases in the abundance of renal tubule Na transporters other than NHE3. Long-term (i.e., 7-day) acid loading with NH4Cl produced large decreases in the abundances of the thiazide-sensitive Na-Cl cotransporter (TSC/NCC) of the distal convoluted tubule and both the β- and γ-subunits of the amiloride-sensitive epithelial Na channel (ENaC) of the collecting duct. In addition, the renal cortical abundance of the proximal type 2 Na-dependent phosphate transporter (NaPi-2) was markedly decreased. In contrast, abundances of the bumetanide-sensitive Na-K-2Cl cotransporter of the thick ascending limb and the α-subunit of ENaC were unchanged. A similar profile of changes was seen with short-term (16-h) acid loading. Long-term (7-day) base loading with NaHCO3resulted in the opposite pattern of response with marked increases in the abundances of the β- and γ-subunits of ENaC and NaPi-2. These adaptations may play critical roles in the maintenance in Na balance when changes in acid-base balance occur.

scholarly journals More actors in ammonia absorption by the thick ascending limb

2012 ◽  
Vol 302 (3) ◽  
pp. F293-F297 ◽  
Author(s):  
Pascal Houillier ◽  
Soline Bourgeois
Keyword(s):  
Current Knowledge ◽  
Collecting Duct ◽  
Ammonia Excretion ◽  
Hydrogen Ion ◽  
Thick Ascending Limb ◽  
Loop Of Henle ◽  
Transport Mechanisms ◽  
Sodium Hydrogen ◽  
Ammonia Transport ◽  
Ammonia Absorption

This review will briefly summarize current knowledge on the basolateral ammonia transport mechanisms in the thick ascending limb (TAL) of the loop of Henle. This segment transports ammonia against a concentration gradient and is responsible for the accumulation of ammonia in the medullary interstitium, which, in turn, favors ammonia secretion across the collecting duct. Experimental data indicate that the sodium/hydrogen ion exchanger isoform 4 (NHE4; Scl9a4) is a sodium/ammonia exchanger and plays a major role in this process. Disruption of murine NHE4 leads to metabolic acidosis with inappropriate urinary ammonia excretion and decreases the ability of the TAL to absorb ammonia and to build the corticopapillary ammonia gradient. However, NHE4 does not account for the entirety of ammonia absorption by the TAL, indicating that, at least, one more transporter is involved.

scholarly journals Inhibition of calcium signaling in descending vasa recta endothelia by ANG II

2000 ◽  
Vol 278 (4) ◽  
pp. H1248-H1255 ◽  
Author(s):  
Thomas L. Pallone ◽  
Erik P. Silldorff ◽  
Zhong Zhang
Keyword(s):  
Collecting Duct ◽  
No Production ◽  
Vascular Bundles ◽  
Thick Ascending Limb ◽  
Ang Ii ◽  
Vasomotor Tone ◽  
Medullary Thick Ascending Limb ◽  
Vasa Recta ◽  
Medullary Collecting Duct ◽  
Peak Phase

The intracellular calcium ([Ca2+]i) response of outer medullary descending vasa recta (OMDVR) endothelia to ANG II was examined in fura 2-loaded vessels. Abluminal ANG II (10− 8 M) caused [Ca2+]i to fall in proportion to the resting [Ca2+]i ( r =0.82) of the endothelium. ANG II (10− 8 M) also inhibited both phases of the [Ca2+]i response generated by bradykinin (BK, 10− 7 M), 835 ± 201 versus 159 ± 30 nM (peak phase) and 169 ± 26 versus 103 ± 14 nM (plateau phase) (means ± SE). Luminal ANG II reduced BK (10− 7 M)-stimulated plateau [Ca2+]i from 180 ± 40 to 134 ± 22 nM without causing vasoconstriction. Abluminal ANG II added to the bath after luminal application further reduced [Ca2+]i to 113 ± 9 nM and constricted the vessels. After thapsigargin (TG) pretreatment, ANG II (10− 8 M) caused [Ca2+]i to fall from 352 ± 149 to 105 ± 37 nM. This effect occurred at a threshold ANG II concentration of 10− 10 M and was maximal at 10− 8 M. ANG II inhibited both the rate of Ca2+ entry into [Ca2+]i-depleted endothelia and the rate of Mn2+ entry into [Ca2+]i-replete endothelia. In contrast, ANG II raised [Ca2+]i in the medullary thick ascending limb and outer medullary collecting duct, increasing [Ca2+]i from baselines of 99 ± 33 and 53 ± 11 to peaks of 200 ± 47 and 65 ± 11 nM, respectively. We conclude that OMDVR endothelia are unlikely to be the source of ANG II-stimulated NO production in the medulla but that interbundle nephrons might release Ca2+-dependent vasodilators to modulate vasomotor tone in vascular bundles.

Regional and segmental localization of AE2 anion exchanger mRNA and protein in rat kidney

1995 ◽  
Vol 269 (4) ◽  
pp. F461-F468 ◽  
Author(s):  
F. C. Brosius ◽  
K. Nguyen ◽  
A. K. Stuart-Tilley ◽  
C. Haller ◽  
J. P. Briggs ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Chloride Concentration ◽  
Transepithelial Transport ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Base Exchange ◽  
Medullary Thick Ascending Limb ◽  
Nephron Segment ◽  
Medullary Collecting Duct

Chloride/base exchange activity has been detected in every mammalian nephron segment in which it has been sought. However, in contrast to the Cl-/HCO3- exchanger AE1 in type A intercalated cells, localization of AE2 within the kidney has not been reported. We therefore studied AE2 expression in rat kidney. AE2 mRNA was present in cortex, outer medulla, and inner medulla. Semiquantitative polymerase chain reaction of cDNA from microdissected tubules revealed AE2 cDNA levels as follows [copies of cDNA derived per mm tubule (+/- SE)]: proximal convoluted tubule, 688 +/- 161; proximal straight tubule, 652 +/- 189; medullary thick ascending limb, 1,378 +/- 226; cortical thick ascending limb, 741 +/- 24; cortical collecting duct, 909 +/- 71; and outer medullary collecting duct, 579 +/- 132. AE2 cDNA was also amplified in thin limbs and in inner medullary collecting duct. AE2 polypeptide was detected in all kidney regions. AE2 mRNA and protein were also detected in several renal cell lines. The data are compatible with the postulated roles of AE2 in maintenance of intracellular pH and chloride concentration and with its possible participation in transepithelial transport.

Dissociation of ammoniagenic enzyme adaptation in rat S1 proximal tubules and ammonium excretion response

1994 ◽  
Vol 267 (3) ◽  
pp. F407-F414 ◽  
Author(s):  
S. R. DiGiovanni ◽  
K. M. Madsen ◽  
A. D. Luther ◽  
M. A. Knepper
Keyword(s):  
Fold Increase ◽  
Proximal Tubules ◽  
Ammonium Excretion ◽  
Production Rates ◽  
Ammonium Production ◽  
Enzyme Adaptation ◽  
Acid Loading ◽  
Microdissected Tubules

We measured ammonium production rates, phosphate-dependent glutaminase (PDG) activity, and glutamate dehydrogenase (GDH) activity in microdissected S1 proximal tubules of rats to investigate the role of adaptations of PDG activity and GDH activity in response to a step increase in acid intake. In vivo ammonium excretion increased much more rapidly than did single-tubule ammonium production in vitro or ammoniagenic enzyme activities measured in microdissected tubules, manifesting an 85-fold increase in the first 24 h. In vitro ammonium production rates in microdissected tubules rose only twofold in the first 24 h, fourfold by day 2, and fivefold by day 4 of acid loading. The adaptation of PDG activity paralleled the increase in single-tubule ammoniagenic capacity measured in vitro. GDH activity, on the other hand, did not change significantly even after 4 days of acid loading. From these observations, we conclude that 1) the adaptation of in vitro ammoniagenic capacity in S1 proximal tubules is temporally associated with an adaptation in PDG activity and not GDH activity, and 2) a major portion of the increased ammonium excretion seen in the first 24 h is due to factors other than an adaptive increase in ammoniagenic enzyme activity.

Cell-specific expression of amiloride-sensitive, Na(+)-conducting ion channels in the kidney

1996 ◽  
Vol 271 (4) ◽  
pp. C1303-C1315 ◽  
Author(s):  
F. Ciampolillo ◽  
D. E. McCoy ◽  
R. B. Green ◽  
K. H. Karlson ◽  
A. Dagenais ◽  
...  
Keyword(s):  
Cell Lines ◽  
Collecting Duct ◽  
Extracellular Fluid ◽  
Cation Channel ◽  
Thick Ascending Limb ◽  
Distal Nephron ◽  
Rt Pcr ◽  
Specific Expression ◽  
Nephron Segments ◽  
Medullary Collecting Duct

Amiloride-sensitive, electrogenic Na+ absorption across the distal nephron plays a vital role in regulating extracellular fluid volume and blood pressure. Recently, two amiloride-sensitive, Na(+)-conducting ion channel cDNAs were cloned. One, an epithelial Na(+)-selective channel (ENaC), is responsible for Na+ absorption throughout the distal nephron. The second, a guanosine 3',5'-cyclic monophosphate (cGMP)-inhibitable cation channel, is conductive to Na+ and Ca2+ and contributes to Na+ absorption across the inner medullary collecting duct (IMCD). As a first step toward understanding the segment-specific contributions(s) of cGMP-gated cation channels and ENaC to Na+ and Ca2+ uptake along the nephron, we used in situ reverse transcription-polymerase chain reaction (RT-PCR) hybridization, solution-phase RT-PCR, and Western blot analysis to examine the nephron and cell-specific expression of these channels in mouse kidney cell lines and/or dissected nephron segments. cGMP-gated cation channel mRNA was detected in proximal tubule, medullary thick ascending limb (mTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and IMCD. cGMP-gated cation channel protein was detected in DCT, CCD, and IMCD cell lines. These observations suggest that hormones that modulate intracellular cGMP levels may regulate Na+, and perhaps Ca2+, uptake throughout the nephron. mRNA for alpha-mENaC, a subunit of the mouse ENaC, was detected in mTAL, DCT, CCD, OMCD, and IMCD. Coexpression of alpha-mENaC and cGMP-gated cation channel mRNAs in mTAL, DCT, CCD, OMCD, and IMCD suggests that both channels may contribute to Na+ absorption in these nephron segments.

scholarly journals Carbonic anhydrase II and IV mRNA in rabbit nephron segments: stimulation during metabolic acidosis

1998 ◽  
Vol 274 (2) ◽  
pp. F259-F267 ◽  
Author(s):  
Shuichi Tsuruoka ◽  
Ann M. Kittelberger ◽  
George J. Schwartz
Keyword(s):  
Metabolic Acidosis ◽  
Carbonic Anhydrase ◽  
Mrna Expression ◽  
Collecting Duct ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Rt Pcr ◽  
Chronic Metabolic Acidosis ◽  
Nephron Segments ◽  
Medullary Collecting Duct

Carbonic anhydrase (CA) facilitates renal bicarbonate reabsorption and acid excretion. Cytosolic CA II catalyzes the buffering of intracellular hydroxyl ions by CO2, whereas membrane-bound CA IV catalyzes the dehydration of carbonic acid generated from the secretion of protons. Although CA II and IV are expressed in rabbit kidney, it is not entirely clear which segments express which isoforms. It was the purpose of this study to characterize the expression of CA II and CA IV mRNAs by specific segments of the nephron using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and to determine the effect of chronic metabolic acidosis on CA expression by those segments. Individual nephron segments (usually 1–2 mm) were isolated by microdissection and subjected to RT-PCR. Amplification was performed simultaneously for CA IV, CA II, and malate dehydrogenase (MDH), a housekeeping gene. The intensities of the PCR products were quantitated by densitometry. CA IV mRNA was expressed by S1 and S2 proximal tubules and by outer medullary collecting duct from inner stripe (OMCDi) and outer stripe and initial inner medullary collecting duct (IMCDi). CA II mRNA was expressed by S1, S2, and S3 proximal tubules, thin descending limb, connecting segment (CNT), and all collecting duct segments. Acid loading induced CA IV mRNA expression in S1 and S2 proximal tubules and in OMCDi and IMCDi. CA II mRNA was induced by acidosis in all three proximal segments and nearly all distal segments beginning with CNT. No upregulation of MDH mRNA expression occurred. These adaptive increases in CA II and IV mRNAs are potentially important in the kidney’s adaptation to chronic metabolic acidosis.

Endothelin synthesis by rabbit renal tubule cells

1991 ◽  
Vol 261 (2) ◽  
pp. F221-F226 ◽  
Author(s):  
D. E. Kohan
Keyword(s):  
High Performance ◽  
Renal Tubule ◽  
De Novo ◽  
Collecting Duct ◽  
Cell Types ◽  
Phospholipid Metabolism ◽  
Thick Ascending Limb ◽  
Medullary Collecting Duct ◽  
Tubule Cells ◽  
Endothelin 3

Endothelins regulate nephron sodium and water transport, prostaglandin E2 (PGE2) synthesis, and phospholipid metabolism. Recent studies suggest that renal tubule cells synthesize endothelins. To determine which nephron sites have such potential, endothelin production by cells derived from different nephron segments was examined. Immunoreactive endothelin 1 (ET-1) and endothelin 3 (ET-3) were measured in supernatants of cultured rabbit proximal tubule (PT), medullary thick ascending limb (MTAL), cortical collecting tubule (CCT), and inner medullary collecting duct (IMCD) cells. All cell types released immunoreactive ET-1 and ET-3. However, the amounts of endothelin produced differed as follows: IMCD greater than MTAL greater than CCT much greater than PT for ET-1 and IMCD greater than MTAL = PT = CCT for ET-3; in all cases ET-1 much greater than ET-3. To confirm de novo ET-3 synthesis, IMCD cells were labeled with [35S]cysteine, and the supernatant was immunoprecipitated with anti-ET-3 antibody. Sample and standard ET-3 eluted at identical positions on high-performance liquid chromatographs, confirming de novo synthesis of ET-3 by cultured IMCD cells. These data raise the possibility of an important functional role for nephron-derived endothelin and, in particular, endothelin produced by tubule cells in the medulla.

Antibody-independent localization of the electroneutral Na+-HCO3− cotransporter NBCn1 (slc4a7) in mice

2008 ◽  
Vol 294 (2) ◽  
pp. C591-C603 ◽  
Author(s):  
Ebbe Boedtkjer ◽  
Jeppe Praetorius ◽  
Ernst-Martin Füchtbauer ◽  
Christian Aalkjaer
Keyword(s):  
Smooth Muscle ◽  
Epithelial Cells ◽  
Smooth Muscle Cells ◽  
Collecting Duct ◽  
Muscle Cells ◽  
Thick Ascending Limb ◽  
Bladder Smooth Muscle ◽  
Kidney Pelvis ◽  
Medullary Collecting Duct ◽  
Cerebellar Purkinje Cells

The expression pattern of the electroneutral Na+-HCO3−cotransporter NBCn1 (slc4a7) was investigated by β-galactosidase staining of mice with a LacZ insertion into the NBCn1 gene. This method is of particular value because it is independent of immunoreactivity. We find that the NBCn1 promoter is active in a number of tissues where NBCn1 has previously been functionally or immunohistochemically identified, including a broad range of blood vessels (vascular smooth muscle cells and endothelial cells), kidney thick ascending limb and medullary collecting duct epithelial cells, the epithelial lining of the kidney pelvis, duodenal enterocytes, choroid plexus epithelial cells, hippocampus, and retina. Kidney corpuscles, colonic mucosa, and nonvascular smooth muscle cells (from the urinary bladder, trachea, gastrointestinal wall, and uterus) were novel areas of promoter activity. Atrial but not ventricular cardiomyocytes were stained. In the brain, distinct layers of the cerebral cortex and cerebellar Purkinje cells were stained as was the dentate nucleus. No staining of skeletal muscle or cortical collecting ducts was observed. RT-PCR analyses confirmed the expression of NBCn1 and β-galactosidase in selected tissues. Disruption of the NBCn1 gene resulted in reduced NBCn1 expression, and in bladder smooth muscle cells, reduced amiloride-insensitive Na+-dependent HCO3− influx was observed. Furthermore, disruption of the NBCn1 gene resulted in a lower intracellular steady-state pH of bladder smooth muscle cells in the presence of CO2/HCO3− but not in its nominal absence. We conclude that NBCn1 has a broad expression profile, supporting previous findings based on immunoreactivity, and suggest several new tissues where NBCn1 may be important.

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