Role of Na+/H+ exchanger NHE3 in nephron function:  micropuncture studies with S3226, an inhibitor of NHE3

Na+/H+ exchanger NHE3 is expressed in the luminal membrane of proximal tubule and thin and thick ascending limb of Henle's loop. To further define its role, the novel NHE3 inhibitor S3226 was employed in micropuncture experiments in nephrons with superficial glomeruli of anesthetized rats. Microperfusion of proximal convoluted tubule with S3226 revealed a dose-dependent inhibition of reabsorption (IC50 of 4–5 μM) with a maximum inhibition of 30% for fluid and Na+. During microperfusion of Henle's loop (last superficial proximal to first superficial distal tubular loop), no effect of S3226 (10 or 30 μM) on the reabsorption of fluid or Na+ was observed. Finally, S3226 (30 μM) left the tubuloglomerular feedback response unaltered as determined by the fall in proximal tubular stop-flow pressure in response to increasing loop of Henle perfusion rate. These studies indicate that NHE3 significantly contributes to fluid and Na+ reabsorption in proximal convoluted tubule. NHE3 appears not to significantly contribute to fluid or Na+reabsorption in the loop of Henle (including the S3 segment of proximal tubule) or macula densa control of nephron filtration.

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Reabsorption and secretion of alpha-ketoglutarate along the rat nephron: a micropuncture study

AJP Renal Physiology ◽

10.1152/ajprenal.1985.248.3.f404 ◽

1985 ◽

Vol 248 (3) ◽

pp. F404-F412 ◽

Cited By ~ 4

Author(s):

B. Ferrier ◽

M. Martin ◽

G. Baverel

Keyword(s):

Proximal Tubule ◽

Progressive Increase ◽

Proximal Convoluted Tubule ◽

Loop Of Henle ◽

Progressive Reduction ◽

Acid Base ◽

Luminal Membrane ◽

Pars Recta ◽

Micropuncture Study ◽

Steady State Levels

The transport of alpha-ketoglutarate (alpha-KG) across the luminal membrane of the rat nephron was studied by micropuncture and microassay techniques. In normal and acidotic rats, approximately 75% of the filtered alpha-KG was reabsorbed in the proximal tubule and 20% in the pars recta and/or loop of Henle at endogenous plasma concentration of alpha-KG. A progressive elevation to steady-state levels of plasma alpha-KG resulted in a progressive reduction of the fractional reabsorption of alpha-KG in the proximal tubule as well as in a progressive increase in the fractional reabsorption of alpha-KG in the pars recta and/or loop of Henle. At plasma alpha-KG concentration 20-40 times above normal, reabsorption of alpha-KG was found to be limited by a maximal tubular capacity. In alkalotic rats, net secretion of alpha-KG in the early proximal convoluted tubule, net reabsorption in the remainder of the proximal convoluted tubule, and net secretion in the pars recta and/or loop of Henle were observed. These micropuncture data indicate that, depending on the acid-base conditions, net reabsorption or net secretion of alpha-KG may occur in at least two distinct sites along the rat nephron.

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Proximal tubule Na+-K+-ATPase activity is inhibited during high-salt diet: evidence for DA-mediated effect

AJP Renal Physiology ◽

10.1152/ajprenal.1988.254.6.f795 ◽

1988 ◽

Vol 254 (6) ◽

pp. F795-F801 ◽

Cited By ~ 15

Author(s):

A. Bertorello ◽

T. Hokfelt ◽

M. Goldstein ◽

A. Aperia

Keyword(s):

Atpase Activity ◽

Proximal Tubule ◽

Significant Inhibition ◽

High Salt ◽

Acid Decarboxylase ◽

Thick Ascending Limb ◽

Amino Acid Decarboxylase ◽

Physiological Importance ◽

Dependent Inhibition ◽

Dose Dependent Inhibition

Locally produced dopamine (DA) causes a reversible and dose-dependent inhibition in Na+-K+-ATPase activity in rat proximal tubule (PT) segments [A. Aperia, A. Bertorello, and I. Seri. Am. J. Physiol. 252 (Renal Fluid Electrolyte Physiol. 21): F32–F45, 1987.]. To examine whether this effect might be of physiological importance, rats were given normal-salt (NS) or high-salt (HS) diet for 10 days. HS diet significantly increased Na excretion but did not alter glomerular filtration rate (GFR). Benserazide (Bz), an inhibitor of the enzyme L-aromatic amino acid decarboxylase (AADC) that converts L-dopa to DA, significantly attenuated the natriuresis in HS rats but had no effect on GFR. By use of immunofluorescence (IF) studies AADC was localized to the PT. Specific AADC IF was not observed in the medulla. In AADC-positive PT segments, Na+-K+-ATPase activity was significantly lower in HS rats than in NS rats (P less than 0.001). In AADC-negative medullary thick ascending limb, Na+-K+-ATPase activity was the same in NS and HS rats. If HS rats were given Bz just before study, PT Na+-K+-ATPase activity increased significantly and was not different from Na+-K+-ATPase activity in PT segments from NS rats. Bz had no significant effect on PT Na+-K+-ATPase activity in NS rats. In PT segments from Bz-treated rats, DA inhibited Na+-K+-ATPase activity already at a dose of 10(-8) M, whereas in segments from NS rats, significant inhibition of Na+-K+-ATPase activity was not observed until DA was increased to 10(-7) M.(ABSTRACT TRUNCATED AT 250 WORDS)

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Micropuncture analysis of single-nephron function in NHE3-deficient mice

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.3.f447 ◽

1999 ◽

Vol 277 (3) ◽

pp. F447-F453 ◽

Cited By ~ 70

Author(s):

John N. Lorenz ◽

Patrick J. Schultheis ◽

Timothy Traynor ◽

Gary E. Shull ◽

Jürgen Schnermann

Keyword(s):

Proximal Tubule ◽

Filtration Rate ◽

Distal Tubule ◽

Transport Processes ◽

Tubuloglomerular Feedback ◽

Thick Ascending Limb ◽

Fluid Reabsorption ◽

Fluid Delivery ◽

Single Nephron ◽

Flow Pressure

The Na/H exchanger isoform 3 (NHE3) is expressed in the proximal tubule and thick ascending limb and contributes to the reabsorption of fluid and electrolytes in these segments. The contribution of NHE3 to fluid reabsorption was assessed by micropuncture in homozygous ( Nhe3 −/−) and heterozygous ( Nhe3 +/−) knockout mice, and in their wild-type (WT, Nhe3 +/+) littermates. Arterial pressure was lower in the Nhe3 −/−mice (89 ± 6 mmHg) compared with Nhe3 +/+ (118 ± 4) and Nhe3 +/−(108 ± 5). Collections from proximal and distal tubules demonstrated that proximal fluid reabsorption was blunted in both Nhe3 +/− and Nhe3 −/−mice (WT, 4.2 ± 0.3; Nhe3 +/−, 3.4 ± 0.2; and Nhe3 −/−, 2.6 ± 0.3 nl/min; P < 0.05). However, distal delivery of fluid was not different among the three groups of mice (WT, 3.3 ± 0.4 nl/min; Nhe3 +/−, 3.3 ± 0.2 nl/min; and Nhe3 −/−, 3.0 ± 0.4 nl/min; P < 0.05). In Nhe3 −/−mice, this compensation was largely attributable to decreased single-nephron glomerular filtration rate (SNGFR): 10.7 ± 0.9 nl/min in the Nhe3 +/+ vs. 6.6 ± 0.8 nl/min in the Nhe3 −/−, measured distally. Proximal-distal SNGFR differences in Nhe3 −/−mice indicated that much of the decrease in SNGFR was due to activation of tubuloglomerular feedback (TGF), and measurements of stop-flow pressure confirmed that TGF is intact in Nhe3 −/−animals. In contrast to Nhe3 −/−mice, normalization of early distal flow rate in Nhe3 +/−mice was not related to decreased SNGFR (9.9 ± 0.7 nl/min), but rather, to increased fluid reabsorption in the loop segment ( Nhe3 +/+, 2.6 ± 0.2; Nhe3 +/−, 3.6 ± 0.5 nl/min). We conclude that NHE3 is a major Na/H exchanger isoform mediating Na+ and fluid reabsorption in the proximal tubule. In animals with NHE3 deficiency, normalization of fluid delivery to the distal tubule is achieved through alterations in filtration rate and/or downstream transport processes.

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Depolarization of the macula densa induces superoxide production via NAD(P)H oxidase

AJP Renal Physiology ◽

10.1152/ajprenal.00515.2006 ◽

2007 ◽

Vol 292 (6) ◽

pp. F1867-F1872 ◽

Cited By ~ 39

Author(s):

Ruisheng Liu ◽

Jeffrey L. Garvin ◽

YiLin Ren ◽

Patrick J. Pagano ◽

Oscar A. Carretero

Keyword(s):

Nitric Oxide ◽

Superoxide Dismutase ◽

Oxidase Inhibitor ◽

Fluorescent Dye ◽

Macula Densa ◽

Superoxide Production ◽

Tubuloglomerular Feedback ◽

Thick Ascending Limb ◽

Loop Of Henle ◽

Nacl Concentration

Superoxide (O2−) enhances tubuloglomerular feedback by scavenging nitric oxide at the macula densa. However, the singling pathway of O2− production in the macula densa is not known. We hypothesized that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces O2− production by the macula densa via NAD(P)H oxidase, which is activated by macula densa depolarization. We isolated and microperfused the thick ascending limb of the loop of Henle and attached macula densa in rabbits. A fluorescent dye, dihydroethidium, was used to detect O2− production at the macula densa. When luminal NaCl was switched from 10 to 80 mM, a situation of initiating maximum tubuloglomerular feedback response, O2− production significantly increased. To make sure that the shifts in the oxyethidium/dihydroethidium ratio were due to changes in O2−, we used tempol (10−4 M), a stable membrane-permeant superoxide dismutase mimetic. With tempol present, when we switched from 10 to 80 mM NaCl, the increase in oxyethidium/dihydroethidium ratio was blocked. To determine the source of O2−, we used the NAD(P)H oxidase inhibitor apocynin. When luminal NaCl was switched from 10 to 80 mM in the presence of apocynin, O2− production was inhibited by 80%. To see whether the effect of increasing luminal NaCl involves Na-K-2Cl cotransporters, we inhibited them with furosemide. When luminal NaCl was switched from 10 to 80 mM in the presence of furosemide, O2− production was blocked. To test whether depolarization of the macula densa induces O2− production, we artificially induced depolarization by adding valinomycin (10−6 M) and 25 mM KCl to the luminal perfusate. Depolarization alone significantly increases O2− production. We conclude that increasing luminal NaCl induces O2− production during tubuloglomerular feedback. O2− generated by the macula densa is primarily derived from NAD(P)H oxidase and is induced by depolarization.

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Potential role of luminal potassium in tubuloglomerular feedback.

Journal of the American Society of Nephrology ◽

10.1681/asn.v8121831 ◽

1997 ◽

Vol 8 (12) ◽

pp. 1831-1837 ◽

Cited By ~ 3

Author(s):

V Vallon ◽

H Osswald ◽

R C Blantz ◽

S Thomson

Keyword(s):

Potential Role ◽

Macula Densa ◽

Tubuloglomerular Feedback ◽

K Channel ◽

Thick Ascending Limb ◽

Retrograde Perfusion ◽

Luminal Membrane ◽

K Concentration ◽

Tubular Flow

Transport through the Na+-2Cl(-)-K+ cotransporter in the luminal membrane of macula densa cells is considered critical for tubuloglomerular feedback (TGF). Although various studies could support the importance of luminal Na+ and Cl-, the role of luminal K+ in TGF has not been thoroughly addressed. The study presented here examines this issue in nephrons with superficial glomeruli of anesthetized male Munich-Wistar-Frömter rats. Ambient Na+ concentration in early distal tubular fluid was approximately 22 mM, suggesting collection sites relatively close to the macula densa segment. First, it was found that ambient early distal tubular K+ concentration is approximately 1.3 mM, i.e., close to the K+ affinity of the Na+-2Cl(-)-K+ cotransporter in the thick ascending limb. Second, it was observed that a change in late proximal tubular flow rate, i.e., a maneuver that is known to induce a TGF response, significantly alters early distal tubular K+ concentration. Third, previous experiments failed to show an inhibition in TGF response during retrograde perfusion of the macula densa with K+-free solutions. Because of a potential K+ influx into the lumen between the perfusion site and the macula densa, however, the K+ channel blocker U37883A was added to the K+-free perfusate. TGF response was assessed as the fall in nephron filtration rate in response to retrograde perfusion of the macula densa segment from early distal tubular site. It was observed that luminal U37883A (100 microM) significantly attenuated TGF. Because adding 5 mM KCl to the perfusate restored TGF in the presence of U37883A and because the inhibitory action of U37883A on tubular K+ secretion was confirmed, the effect of U37883A on TGF was most likely caused by inhibition of K+ influx into the perfused segment, which decreased luminal K+ concentration at the macula densa. The present findings support a potential role for luminal K+ in TGF, which is in accordance with a transmission of the TGF signal across the macula densa via Na+-2Cl(-)-K+ cotransporter.

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Renal effects of endogenous prostaglandin synthesis promoter ONO-3122

AJP Renal Physiology ◽

10.1152/ajprenal.1992.262.6.f1047 ◽

1992 ◽

Vol 262 (6) ◽

pp. F1047-F1054

Author(s):

T. Takabatake ◽

H. Hara ◽

Y. Ishida ◽

H. Ohta ◽

K. Kobayashi

Keyword(s):

Proximal Tubule ◽

Prostaglandin Synthesis ◽

Fluid Replacement ◽

Tubuloglomerular Feedback ◽

Loop Of Henle ◽

Distal Nephron ◽

Water Reabsorption ◽

Renal Vasculature ◽

Renal Effects ◽

Collecting Tubules

The renal effects of a prostaglandin synthesis agonist, 1-iodo-3-aminomethyl-5,6,7,8-tetrahydro-2-naphthol (ONO-3122), were investigated in anesthetized rats. ONO-3122 (0.3 mg/kg + 0.3 mg.kg-1.h-1 iv) doubled the urinary excretion of the main metabolites of prostaglandin F, and induced transient increases in renal blood flow and glomerular filtration rate (GFR) with a marked, stable natriuresis. Indomethacin suppressed the natriuresis. When the diuretic fluid losses were replaced, micropuncture showed an unaltered reabsorption of sodium in the proximal tubule but reductions in the loop of Henle (86 +/- 1 vs. 76 +/- 1%) and in the more distal segments (98 +/- 1 vs. 83 +/- 3%) with comparable reductions in water reabsorption. Potassium secretion was seen in the distal and collecting tubules. Without fluid replacement, sodium reabsorption was reduced in the loop and more distal nephron but increased in the proximal tubule. Differences between proximal and distal nephron GFR were unaffected by systemic ONO-3122. Loop perfusion with ONO-3122 did not change tubuloglomerular feedback responses, which were, however, completely suppressed by furosemide. It is concluded that ONO-3122 stimulates renal prostaglandin biosynthesis, transiently dilates renal vasculature, and induces natriuresis mainly by suppressing sodium and water reabsorption in the loop of Henle and the more distal nephron. Luminal ONO-3122 does not affect the tubuloglomerular feedback.

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Studies of the urinary acidification defect induced by lithium

AJP Renal Physiology ◽

10.1152/ajprenal.1982.242.1.f23 ◽

1982 ◽

Vol 242 (1) ◽

pp. F23-F29 ◽

Cited By ~ 2

Author(s):

N. Bank ◽

P. D. Lief ◽

H. S. Aynedjian ◽

B. F. Mutz

Keyword(s):

Proximal Tubule ◽

Renal Tubular Acidosis ◽

Electrical Potential ◽

Distal Renal Tubular Acidosis ◽

Distal Nephron ◽

Luminal Membrane ◽

Proximal Tubular ◽

Renal Tubular ◽

Convoluted Tubules

Experiments were carried out in rats and isolated turtle bladders to study the defect in H+ transport induced by LiCl. After 3-4 days of intraperitoneal LiCl, rats developed urinary findings of "distal" renal tubular acidosis. Proximal tubular fluid pH measured in situ by glass microelectrodes was higher in lithium-treated rats than in acidotic controls. Proximal fluid total CO2 [tCO2] was also higher, and the fraction of tCO2 leaving the proximal tubule was 14 vs. 7% (P less than 0.001). Impaired acidification was also apparent beyond distal convoluted tubules, as judged by normal distal tCO2 reabsorption but increased HCO3(-) in the urine. During NaHCO3 loading, the proximal defect was ameliorated but not the distal. Turtle bladder studies showed that mucosal lithium inhibits H+ secretion secondary to reducing transepithelial electrical potential, presumably by hyperpolarization of the luminal membrane. A similar mechanism may be responsible for lithium's effect on the distal nephron. Inhibition of proximal tubular HCO3(-) reabsorption is probably not attributable to electrical potential changes but might be due to interference of luminal membrane Na+ entry by Li+ and reduced (Na+ + Li+)-H+ exchange.

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ANG II is a mitogen for a murine cell line isolated from medullary thick ascending limb of Henle's loop

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.5.f940 ◽

1995 ◽

Vol 268 (5) ◽

pp. F940-F947 ◽

Cited By ~ 7

Author(s):

G. Wolf ◽

F. N. Ziyadeh ◽

U. Helmchen ◽

G. Zahner ◽

R. Schroeder ◽

...

Keyword(s):

Receptor Antagonist ◽

Cell Line ◽

Thick Ascending Limb ◽

Dependent Manner ◽

Ang Ii ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Medullary Thick Ascending Limb ◽

Henle's Loop ◽

Proximal Tubular

A murine SV40-transformed renal epithelial cell line derived from medullary thick ascending limb of Henle's loop (MTAL) was established and characterized by morphology, antigen expression, and biochemical criteria. These MTAL cells express a single class of high-affinity receptors for angiotensin II (ANG II) and transcripts for the AT1 subtype of ANG II receptors. ANG II, in a dose-dependent manner, induced proliferation of MTAL cells. This observation is in striking contrast to syngeneic proximal tubular cells in which it was previously shown that the peptide induced cellular hypertrophy and slightly inhibited proliferation [G. Wolf and E. G. Neilson. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28: F768-F777, 1990]. The AT1-receptor antagonist losartan (10(-6) M), but not an AT2-receptor antagonist, blocked the mitogenic effects of ANG II in MTAL cells. Coincubation of quiescent MTAL cells with ANG II and 5% fetal calf serum further increased proliferation compared with cells grown only in serum. In contrast to proximal tubular cells, ANG II failed to induce transforming growth factor-beta 1 mRNA and protein synthesis in MTAL cells. Our data collectively suggest that ANG II is a mitogen for MTAL cells in vitro. Therefore, epithelial cells derived from different parts of the nephron, even when transformed with SV40 virus and while under cell culture conditions, exhibit a distinct pattern of growth behavior after stimulation with ANG II.

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Osmolality, bicarbonate concentration, and water reabsorption in proximal tubule of the dog nephron

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1963.205.2.273 ◽

1963 ◽

Vol 205 (2) ◽

pp. 273-280 ◽

Cited By ~ 46

Author(s):

James R. Clapp ◽

John F. Watson ◽

Robert W. Berliner

Keyword(s):

Plasma Concentration ◽

Proximal Tubule ◽

Proximal Convoluted Tubule ◽

Water Reabsorption ◽

Bicarbonate Concentration ◽

Water Diuresis ◽

Vasopressin Infusion ◽

Proximal Tubular

Micropuncture and microanalytical techniques were used to study the effect of antidiuresis and water diuresis on osmolality, bicarbonate concentration, and water reabsorption in the proximal tubule of the dog nephron. Samples collected during antidiuresis and water diuresis remained isotonic to plasma throughout the first 50% of the proximal convoluted tubule. Mean bicarbonate concentrations of 16 mEq/liter and 17 mEq/liter were found in the middle third of the tubule during antidiuresis and water diuresis, respectively. These values were slightly less than the plasma concentration of 22 mEq/liter. Proximal tubular fluid samples for inulin concentration were collected during antidiuresis, water diuresis, and during vasopressin infusion in water-loaded dogs. A mean tubular fluid to plasma (TF/P) inulin ratio of 2.3 was found in the middle third of the proximal tubule during antidiuresis. This value is significantly different ( P < 0.01) from a mean of 1.6 in the same portion of the tubule during water diuresis. Vasopressin administration to hydrated dogs returned the TF/P inulin ratio in the middle third of the proximal tubule to 2.0. These results suggest that vasopressin stimulated Na reabsorption in the proximal tubule of the dog nephron.

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