Luminal flow induces eNOS activation and translocation in the rat thick ascending limb.
ABSTRACT Nitric oxide (NO) produced by endothelial NO synthase (eNOS) acts as an autacoid to inhibit NaCl absorption in the thick ascending limb of the loop of Henle (THAL). In the vasculature, shear stress activates eNOS. We hypothesized that increasing luminal flow activates eNOS and enhances NO production in the THAL. We measured NO production by isolated, perfused THALs using a NO-sensitive microelectrode. Increasing luminal flow from 0 to 20 nl/min increased NO production by 43.1 +/- 4.1 pA/mm of tubule (n = 10, P < 0.05), and this response was blunted (92%) by the NOS inhibitor L-(omega)nitro-methylarginine (P < 0.05). We studied the effect of flow on eNOS subcellular localization. In the absence of flow, eNOS was diffusely localized throughout the cell (basolateral = 33 +/- 4%; middle = 27 +/- 3%; apical = 40 +/- 4% of total eNOS). Increasing luminal flow induced eNOS translocation to the apical membrane, as evidenced by a 60% increase in eNOS immunoreactivity in the apical membrane (from 40 +/- 4 to 65 +/- 2%; n = 6; P < 0.05). Disrupting the actin cytoskeleton with cytochalasin D (10 microM) reduced flow-induced NO production by 62% (from 37.1 +/- 3.4 to 14.0 +/- 2.4 pA/mm tubule, n = 7, P < 0.04) and blocked flow-induced eNOS translocation. Flow also increased the amount of phosphorylated eNOS (Ser1179) at the apical membrane (from 25 +/- 2 to 56 +/- 2%; P < 0.05). We conclude that increasing luminal flow induces eNOS activation and translocation to the apical membrane in THALs. These are the first data showing that flow regulates eNOS in epithelial cells. This may be an important mechanism for regulation of NO levels in the renal medulla.
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ABSTRACT: The involvement of purinergic signalling in kidney physiology and pathophysiology is rapidly gaining recognition and this is a comprehensive review of early and recent publications in the field. Purinergic signalling involvement is described in several important intrarenal regulatory mechanisms, including tuboglomerular feedback, the autoregulatory response of the glomerular and extraglomerular microcirculation and the control of renin release. Furthermore, purinergic signalling influences water and electrolyte transport in all segments of the renal tubule. Reports about purine- and pyrimidine-mediated actions in diseases of the kidney, including polycystic kidney disease, nephritis, diabetes, hypertension and nephrotoxicant injury are covered and possible purinergic therapeutic strategies discussed.Purinergic Signalling 11/2013; · 2.64 Impact Factor
Article: Niere, Kochsalz und BlutdruckWiener Medizinische Wochenschrift 07/2008; 158:365-369.
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ABSTRACT: Pathways that contribute to TNF production by the kidney are not well defined. Mice given 1% NaCl in the drinking water for 3 days exhibited a 2.5-fold increase in urinary, but not plasma, TNF levels compared with mice given tap water. Since furosemide attenuated the increase in TNF levels, we hypothesized that hypertonic NaCl intake increases renal TNF production by a pathway involving NKCC2. A 2.5-fold increase in NKCC2A mRNA accumulation was observed in medullary thick ascending limb (mTAL) tubules from mice given 1% NaCl; a concomitant 2-fold increase in NFAT5 mRNA and protein expression was observed in outer medulla. Urinary TNF levels were reduced in mice given 1% NaCl after an intrarenal injection of a lentivirus construct designed to specifically knockdown NKCC2A (EGFP-N2A-ex4); plasma levels of TNF did not change after injection of EGFP-N2A-ex4. Intrarenal injection of EGFP-N2A-ex4 also inhibited the increase of NFAT5 mRNA abundance in outer medulla of mice given 1% NaCl. TNF production by primary cultures of mTAL cells increased approximately 6-fold in response to an increase in osmolality to 400 mosmol/kg H2O produced with NaCl, and was inhibited in cells transiently transfected with a dnNFAT5 construct. Transduction of cells with EGFP-N2A-ex4 also prevented increases in TNF mRNA and protein production in response to high NaCl concentration and reduced transcriptional activity of a NFAT5 promoter construct. Since NKCC2A expression is restricted to the TAL, NKCC2A-dependent activation of NFAT5 is part of a pathway by which the TAL produces TNF in response to hypertonic NaCl intake.AJP Renal Physiology 12/2012; · 4.42 Impact Factor
Luminal Flow Induces eNOS Activation and Translocation in the
Rat Thick Ascending Limb I
Pablo A. Ortiz*, Nancy J. Hong, and Jeffrey L. Garvin
*Corresponding Author: Pablo A. Ortiz, Ph.D.
Division of Hypertension and Vascular Research
Department of Internal Medicine
Henry Ford Hospital
2799 W. Grand Boulevard
Detroit, MI 48202
Phone: (313) 916-8501
Fax: (313) 916-1479
Running title: Flow-induced NO production in the THAL
Articles in PresS. Am J Physiol Renal Physiol (April 6, 2004). 10.1152/ajprenal.00382.2003
Copyright © 2004 by the American Physiological Society.
NO produced by endothelial NO synthase (eNOS) acts as an autacoid to inhibit NaCl absorption
in the thick ascending limb of the loop of Henle (THAL). Little is known about regulation of NO
production and eNOS activity in this nephron segment. In the vasculature, shear stress increases
NO production via activation of eNOS. We hypothesized that increasing luminal flow activates
eNOS and enhances NO production in the THAL. We first measured the effect of flow on NO
production by isolated perfused THALs using a NO-sensitive microelectrode. We found that
increasing luminal flow from 0 to 20-25 nl/min increased NO production by 43.1 ± 4.1 pA/mm
of tubule length (n = 10, p < 0.05). The NOS inhibitor L-NAME (5 mM), blunted flow-induced
NO production by 92% (n = 6; p < 0.05). Since eNOS changes its localization when activated in
endothelial cells, we studied the effect of flow in eNOS subcellular localization. In the absence
of luminal flow, eNOS was diffusely localized throughout the cell (basolateral = 33±4%; middle
= 27±3%; apical = 40±4% of total eNOS immunofluorescence; n = 6). Increasing luminal flow
to 20-25 nl/min induced eNOS translocation to the apical membrane, as evidenced by a 60%
increase in eNOS immunoreactive protein in the apical membrane (from 40±4% to 65±2%; n =
6; p < 0.05). We next tested whether disruption of the cytoskeleton could block flow-induced NO
production and eNOS translocation. We found that pretreatment of THALs with cytochalasin D
(10 µM) reduced flow-induced NO production by 62% (from 37.1 ± 3.4 to 14.0 ± 2.4 pA/mm
tubule length, n = 7, p<0.04) and blocked flow-induced eNOS translocation to the apical
membrane (basolateral = 49±3%; middle = 25±1%; apical = 26±2%; n = 6). Finally, we tested
whether flow induces phosphorylation of eNOS at the apical membrane using a phospho-specific
antibody. Flow increased the amount of phosphorylated eNOS (ser 1179) in the apical membrane
(from 25 ± 2% to 56 ± 2%; p<0.05). We concluded that increasing luminal flow activates eNOS
and induces its translocation to the apical membrane in the THAL. We believe these are the first
data showing that flow regulates eNOS in epithelial cells. Because luminal flow in the THAL
may be affected by various conditions, this may be an important mechanism for overall
regulation of NO levels in the renal medulla.
Key words: thick ascending limb, nitric oxide, trafficking, NOS III, epithelial cells
Nitric oxide (NO) plays an important role in regulation of salt and water reabsorption by
the kidney (31). NO exerts portent natriuretic and diuretic effects in the kidney in part by directly
inhibiting renal tubular transport (15,16,31,32). We have previously observed that endogenously
produced NO acts as an autacoid in the thick ascending limb of the loop of Henle (THAL)
inhibiting net NaCl reabsorption by decreasing apical Na/K/2Cl cotransport (27,36). We recently
identified eNOS as the NOS isoform responsible for NO production and regulation of NaCl
absorption by the THAL (33,35). However, despite the importance of eNOS in the THAL, little
is known about the factors that regulate eNOS and NO production along the nephron.
In cultured inner medullary collecting duct cells, increasing parallel flow on the apical
side heightened nitrite production, suggesting that flow stimulated NO production by these renal
epithelial cells (4). In endothelial cells lining the blood vessels, flow-induced shear stress is one
of the most potent agonists for eNOS activation and NO production (11,14,42). In these cells,
eNOS is localized mainly to the Golgi apparatus and plasma membrane caveolae (13). eNOS
agonists such as vascular endothelial growth factor (VEGF), bradykinin and estradiol have been
reported to induce changes in its subcellular localization (10,18,37). However, it is not known
whether luminal flow stimulates eNOS in the THAL, or whether the subcellular localization of
eNOS can be altered in renal or other epithelial cells.
Similar to endothelial cells, epithelial cells of the THAL are arranged in tubular structures
and are subjected to changes in luminal flow. However, little is known about the role of luminal
flow in regulating eNOS activity and NO production in renal tubules, or whether this occurs by a
mechanism similar to that observed in endothelial cells. We hypothesized that flow induces
eNOS activation and translocation in the THAL. By measuring NO production and subcellular
localization of eNOS in isolated THALs, we found that increasing luminal flow stimulated NO
production and induced translocation of eNOS from the basolateral membrane and cytoplasm to
the apical membrane of THALs. Both activation and translocation were dependent on an intact
actin cytoskeleton. We concluded that flow stimulates eNOS activity and induces its
translocation in the THAL.
Isolation and Perfusion of THALs
Male Sprague-Dawley rats weighing 120 to 150 g (Charles River Breeding Laboratories,
Wilmington, MA) were fed a diet containing 0.22% sodium and 1.1% potassium (Purina,
Richmond, IN) for at least 5 days. On the day of the experiment, rats were anesthetized with
ketamine (100 mg/kg body wt i.p.) and xylazine (20 mg/kg body wt i.p.). After anesthesia, the
abdominal cavity was opened, and the left kidney was bathed in ice-cold saline and removed.
Coronal slices were placed in oxygenated physiological saline (130 mM NaCl, 4 mM KCl, 2.5
mM NaH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 6.0 mM alanine, 2.0 mM calcium lactate, 1.0
mM sodium citrate, 10 mM HEPES pH 7.4 with NaOH). THALs were dissected under a
stereomicroscope from the medullary rays at 4-10°C and transferred to a temperature-regulated
chamber and held between concentric glass pipettes at 37 ± 1°C. When luminal flow was
desired, THALs were perfused at a maximum rate of >50 nl/min or at physiological flow rates of
20-25 nl/min using a nanoliter syringe pump (Harvard Apparatus, Holliston, MA).
NO release by isolated THALs
NO released by THALs was measured using an amperometric microelectrode selective
for NO (inNO measuring system, Harvard Apparatus) as described previously (30). For this we