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作者：Pablo A. Ortiz, Nancy J. Hong, and Jeffrey L. Garvin作者单位：Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202 + G' b( @& \+ m5 u
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【摘要】* s0 e& E* C: B" D* d% K5 K6 r. B
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 - 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 µM) 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.
5 X1 p w& z3 Y* ?) [# H8 x 【关键词】 nitric oxide trafficking nitric oxide synthase III epithelial cells. @# t, F* A8 `; a6 Q! v
NITRIC OXIDE ( NO ) PLAYS AN important role in regulation of salt and water reabsorption by the kidney ( 31 ). NO exerts potent 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 endothelial NO synthase (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.
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In cultured inner medullary collecting duct cells, increasing parallel flow on the apical side heightened nitrite production, suggesting that flow stimulates 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.
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0 w* R, U$ X0 O" _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.
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/ J1 l( G: |; q& Z0 B8 yMETHODS
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Isolation and perfusion of THALs. Male Sprague-Dawley rats weighing 120-150 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 ip) and xylazine (20 mg/kg body wt ip). 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 (in mM: 130 NaCl, 4 KCl, 2.5 NaH 2 PO 4, 1.2 MgSO 4, 5.5 glucose, 6.0 alanine, 2.0 calcium lactate, 1.0 sodium citrate, and 10 HEPES, pH 7.4, titrated with NaOH). THALs were dissected from the medullary rays under a stereomicroscope 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 50 nl/min or at physiological flow rates of 20-25 nl/min using a nanoliter syringe pump (Harvard Apparatus, Holliston, MA).+ @& k! g) E- k, m' R: m( S P: K1 {
. B% p( [5 J9 p! Y/ yAll protocols involving animals were approved by the Institutional Animal Care and Use Committee at Henry Ford Hospital.
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: x3 ? E5 {# RNO 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 used a special microperfusion setup designed to reduce ambient electrical noise and provide precise temperature control in the perfusion chamber. The chamber was surrounded by a circulating water bath connected to a 10-gallon reservoir and mounted on an inverted microscope (Nikon Diaphot). The microscope was surrounded by a Faraday cage, which was insulated to prevent fluctuations in ambient temperature that could affect the electrical signal. The setup was placed in an isolated room where temperature and ambient humidity were controlled. The bath used for tubule perfusion had a volume of 2.5 ml and was not exchanged during the experimental period (90 min). The NO sensor was connected to a picoammeter equipped with an internal 24-bit analog-to-digital converter and digital filters (inNO measuring system, Harvard). Data output was recorded, played back, and analyzed using inNO software. The bath and luminal solution contained (in mM) 8 Na 2 HPO 4, 0.2 NaHCO 3, 130 NaCl, 4 KCl, 1.4 CaCl 2, 1 MgSO 4, 1 sodium acetate, 5.5 glucose, 0.1 L -arginine, and 6 L -alanine. Solutions were gassed with air before the experiments, and pH was adjusted to 7.4. THALs were placed in contact with the electrode so that most of the tubule would touch the sensor. The positions of the electrode and tubule were monitored by a CCD camera. After a 30-min equilibration period in which a stable baseline was achieved (±5 pA), NO production was measured for 10 min and averaged as basal NO production. Then flow was started, and NO release was monitored continuously until it reached a new level, at which point a new 10-min period was measured and averaged. The difference between baseline and the level reached after luminal flow was increased was taken as the response and normalized per tubule length (mm) as described previously ( 30 ). When the effect of L -nitro- L -arginine methyl ester ( L -NAME) or cytochalasin D on NO production was tested, the drugs were added to the bath at the beginning of the experiment.! s( J# N9 T' {# n
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Immunodetection of eNOS and phosphorylated eNOS in isolated thick ascending limbs using confocal microscopy. Tubules were fixed in the absence or presence of luminal flow (20-25 nl/min) for 15 min with 4% paraformaldehyde in 150 mM NaCl and 10 mM Na 2 HPO 4, pH 7.4. Fixed cells were blocked for 30 min with 1% BSA in TBS-T perfused into the lumen followed by a 30-min incubation with primary antibodies diluted in 1% BSA in TBS-T [eNOS, phosphorylated (P)-eNOS 1:1,000] in the lumen. Cells were washed with TBS-T for 5 min in both lumen and bath and then incubated for 30 min with a secondary antibody conjugated to a fluorescent dye (Alexa Fluor 488 goat anti-mouse IgG) diluted 1:200 in 1% BSA in TBS-T. Cells were then washed for 5 min with TBS-T in lumen and bath, and fluorescent images were acquired using a confocal microscope. All drugs were added to the bath before the experiment. Monoclonal antibodies to eNOS were obtained from BD Transduction Labs (San Diego, CA); anti-P-eNOS (Ser1179) was obtained from Cell Signaling Technologies (Beverly, MA). Alexa Fluor 488 goat anti-mouse IgG was from Molecular Probes (Eugene, OR).1 V, |' h; b. ~/ W
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An Oz with Intervision confocal laser-scanning system was used (488-nm excitation and 500-nm low-pass filter, 30% laser power, 25-µm slit, 1912 brightness setting, 2978 contrast setting; T series protocol with 1 image acquired for 2.133-s duration, normal scan, medium resolution, 100-ns sample time, and jump average with n = 64 images; Noran Instruments, Middleton, WI). Two-dimensional image analysis was performed with Intervision software. Briefly, a straight line was drawn across a single cell, and a pixel-intensity histogram was obtained. Fluorescent intensities were obtained from 1 ) a point in the apical membrane, 2 ) a point in the basolateral membrane, and 3 ) a point in the middle of the cell equidistant from both membranes. The intensities in the three regions (apical, middle, and basolateral) were added, and the percent intensity for eNOS or P-eNOS in the three regions was calculated. In each tubule, three cells from different regions (proximal, midway, and distal from the perfused side) were analyzed. Because we observed that the intracellular distribution of eNOS and changes in the presence of flow were similar in all cells along a tubule, the intensity values of cells in these three regions were pooled. Image files were converted to.TIFF via SGI imaging software and pseudocolored with Adobe Photoshop.9 o8 `2 }9 {' D9 h3 v* |
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Statistics. Results are expressed as means ± SE. Student's paired t -test was used to determine statistical differences between means before and after treatment in the same group of tubules. One-way ANOVA was used to determine statistical differences between different groups, and P
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We first tested whether increasing luminal flow could heighten NO production by isolated THALs. A maximal increase in luminal 50 nl/min) increased the NO signal significantly, averaging 91.8 ± 30.9 pA/mm of tubule length ( n = 8, P & @. c9 B+ J' F! T I: i |
6 p- V# R" T' D! f9 i+ ?& T* oFig. 1. Flow induces nitric oxide (NO) production by isolated thick ascending limbs of Henle's loop (THALs). A : representative trace of flow-induced NO production by an isolated THAL is shown. B 50 nl/min produced an average NO response of 91.8 ± 30.9 pA/mm of tubule length ( n = 8, P
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9 P6 Z: i. }6 F1 t0 W2 GTo make sure the increase in the electrode signal was due to NO produced by THALs and not an artifact, we tested whether we could block the increase in NO production with a NOS inhibitor. THALs were preincubated with 5 mM L -NAME for 30 min, and then flow was increased. We observed that L -NAME blunted the response to flow by 92% (from 91.8 ± 30.9 to 10.1 ± 2.2 pA/mm of tubule, n = 6, P $ a8 o0 |, ^9 l* {% C8 _
+ d, t p4 K& O# i1 jFig. 2. NO synthase (NOS) inhibition with L -nitro- L -arginine methyl ester ( L -NAME) blocks flow-induced NO production by isolated THALs. Under control conditions, increasing luminal flow from 0 to 4 e; \% N$ I. h2 E; @+ @
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Because eNOS activation is associated with changes in eNOS localization, we studied the effect of luminal flow on eNOS localization in isolated THALs. In the absence of flow, most eNOS immunostaining was diffusely distributed throughout the cell cytoplasm and apical and basolateral membranes (apical membrane = 40 ± 2%, middle= 27 ± 2%, basolateral membrane = 33 ± 3% of total fluorescence intensity, n = 6) ( Fig. 3 ). We then studied whether increasing luminal flow from 0 to 20-25 nl/min could induce redistribution of eNOS. Thirty minutes after flow was increased, eNOS translocated from the basolateral membrane and cytoplasm to the apical membrane, which was demonstrated by a significant increase in eNOS at the apical membrane (from 40 ± 2 to 64 ± 2%, n = 6, P G, n$ W! ], t* r7 S) Y
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Fig. 3. Flow induces endothelial (e)NOS translocation to the apical membrane in THALs. A and B : representative confocal micrographs of eNOS localization in isolated THALs in the absence ( A : NO FLOW) and presence of 20-25 nl/min luminal flow ( B : FLOW) for 30 min (scale bar = 10 µm). C : relative amounts of eNOS immunolabeling in the basolateral membrane, the middle of the cell, and the apical membrane of THALs. Increasing luminal flow from 0 to 20-25 nl/min significantly increased eNOS at the apical membrane of THALs ( n = 6/protocol, * P ! K) W$ @2 i$ S* V
# J& R6 ~$ F* _9 yThe actin cytoskeleton is involved in mechanotransduction ( 5, 28 ) and is necessary for flow-induced NO production in endothelial cells ( 22 ). Thus we tested whether we could block flow-induced NO production by THALs with cytochalasin D, an agent that destabilizes the actin cytoskeleton ( 2, 17 ). THALs were preincubated with 10 µM cytochalasin D for 30 min in the absence of flow, and then luminal flow was increased to 20-25 nl/min. Cytochalasin D blunted the response to flow by 62% compared with untreated THALs (from 37.1 ± 3.4 to 14.0 ± 2.4 pA/mm, n = 7, P
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Fig. 4. Cytochalasin D blocks flow-induced NO production by isolated THALs. Under control conditions, increasing luminal flow from 0 to 20-25 nl/min produced an average NO response of 37.1 ± 3.4 pA/mm of tubule length ( n = 6, P [: a- L9 X5 |
/ M" e, L7 j; @4 lBecause cytochalasin D blocked flow-induced NO production, we next tested whether we could inhibit eNOS translocation by disrupting the cytoskeleton. We found that preincubation of THALs with cytochalasin D (10 µM) blocked flow-induced eNOS translocation to the apical membrane (flow: apical = 64 ± 2% vs. flow cytochalasin D: apical = 26 ± 2%, n = 6). The amount of eNOS in the cytoplasm did not change (flow: middle = 18 ± 2% vs. flow cytochalasin D: middle = 25 ± 1%, n = 6), whereas the amount of eNOS in the basolateral membrane was increased from 17 ± 1 to 49 ± 3% ( n = 6, P ! x! v Y/ a0 Q T3 s5 ]
& T/ `) m/ k7 D" s# yFig. 5. Cytochalasin D blocks flow-induced eNOS translocation in isolated THALs. A and B : representative confocal micrographs of eNOS localization in isolated THALs after 30 min of 20-25 nl/min luminal flow in the absence ( A ) and presence ( B ) of cytochalasin D (1 µM) (scale bar = 10 µm). C : effect of luminal flow on relative eNOS immunolabeling in the basolateral membrane, the middle of the cell, and the apical membrane of THALs in the absence and presence of cytochalasin D ( n = 6/protocol, * P
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In endothelial cells, activation of eNOS by flow is dependent on eNOS phosphorylation at the Ser1179 residue ( 6, 11 ). To make sure flow activates eNOS, which then translocates to the apical membrane, we measured the effect of flow on the amount of P-eNOS by immunofluorescence and confocal microscopy. We found that 30 min after luminal flow was initiated, the amount of P-eNOS (Ser1179) in the apical membrane increased from 25 ± 2 to 56 ± 2% of total P-eNOS, a 124% increase, as detected with a phospho-specific antibody ( n = 4, P
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2 \" }/ l; J. l7 I8 sFig. 6. Effect of luminal flow (20-25 nl/min) on relative amounts of phosphorylated (P)-eNOS (Ser1179) immunolabeling at the apical membrane of THALs ( n = 6/protocol, * P
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We found that increasing luminal flow induced NO production by rat THALs. Flow-induced NO production was almost completely blocked by L -NAME, indicating that NOS is involved in NO production due to increased flow. Luminal flow also induced eNOS translocation from the basolateral membrane and cytoplasm to the apical membrane of freshly isolated THALs. The cytoskeleton-destabilizing agent cytochalasin D significantly blunted flow-induced NO production and blocked flow-induced eNOS translocation to the apical membrane. Finally, luminal flow increased the amount of eNOS phosphorylated at the Ser1179 residue in the apical membrane. We concluded that luminal flow stimulates eNOS activity and induces translocation of eNOS to the apical membrane of THALs. We believe these are the first data showing that flow induces eNOS translocation and activation in epithelial cells of the kidney or other tissues. Given the importance of eNOS-derived NO in the regulation of various physiological processes in epithelial cells of the kidney ( 33, 35, 49 ), respiratory tract ( 50 ) and testis ( 51 ), this mechanism could be essential to the regulation of NO levels in these cells, which are subjected to changes in luminal flow.
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In the vasculature, flow-induced shear stress has been shown to be the most potent agonist of NO production by endothelial cells, and the molecular mechanisms leading to NO production in these cells have been studied in depth ( 11, 14, 42 ). However, despite the importance of NO in regulating THAL function, it is not known whether flow regulates NO production in this nephron segment. We first studied whether a large increase in luminal 50 nl/min) could produce a measurable NO response and found that this maneuver readily increased the response from the NO-sensitive microelectrode. To test whether physiological flow rates could also stimulate NO release, we increased luminal flow from 0 to 20-25 nl/min. We found that increasing luminal flow rates to this level also stimulated NO production, with the average response being roughly one-half of that obtained with high flow rates. To ensure that the response caused by increased luminal flow was due to NO produced by NOS and not an artifact, we tested whether it could be blocked by the NOS inhibitor L -NAME. We found that L -NAME completely suppressed the increase in NO caused by flow, indicating that this increase was due to activation of NOS. Because we have previously shown that NO production by THALs is dependent on L -arginine, the substrate for NOS ( 30 ), these experiments were carried out with a physiological concentration of L -arginine (100 µM) in the bath and luminal solutions. Taken together, our data indicate that increasing the luminal flow rate stimulates NO production by the THAL.5 ~1 e7 @1 K. N( [& ]4 c; O# \, O6 {
- o2 ^ J D" Q# O: P) OWe have previously shown that eNOS is the NOS isoform that mediates NO production and regulates NaCl absorption by the THAL ( 33, 35 ). In endothelial cells, activation of eNOS by most agonists alters the intracellular localization of the enzyme ( 10, 18, 37 ). Thus we studied whether flow could change eNOS localization in the THAL. We found that increasing luminal flow from 0 to 20-25 nl/min induced eNOS translocation from the basolateral membrane and cytoplasm to the apical membrane. Although flow-induced shear stress has been shown to be the most potent agonist of eNOS in vascular endothelial cells, it is not clear whether it induces eNOS translocation in these cells. Thus we believe these are the first data to show that luminal flow induces eNOS translocation between different subcellular compartments in mammalian cells. In endothelial cells, some agonists induce eNOS translocation from the plasma membrane to the cytoplasm and dissociation of eNOS from caveolin-1 ( 18, 37 ). However, the mechanism for eNOS translocation we observed in the THAL is apparently different from endothelial cells, in which eNOS translocates from one side of the cell (basolateral membrane) and from the cytoplasm to the apical membrane. Thus the mechanism by which eNOS trafficking is regulated appears to be cell type specific.3 i+ U6 [7 s# @8 U
0 b; k/ e$ j* q/ cTo learn how flow induces eNOS activation in THALs, we studied whether the actin cytoskeleton is involved in this event. This is important for two reasons: 1 ) the cytoskeleton has been shown to be involved in sensing and transmitting signals caused by mechanical stimuli in most cells ( 5, 34 ) and 2 ) it has been shown that an intact actin cytoskeleton is required for flow-induced NO production in endothelial cells ( 22 ). Our data showed that cytochalasin D, an agent that destabilizes actin fibers, blunted flow-induced NO production by THALs by 62%, and it also blocked flow-induced eNOS translocation to the apical membrane but had no effect on eNOS localization in the absence of luminal flow. While these data suggest that the actin cytoskeleton plays a role in the response initiated by flow, it could either sense changes in flow, acting as the initiator of the signaling cascade, or merely mediate the signaling leading to eNOS activation. In the proximal tubule, disruption of the cytoskeleton with cytochalasin D blocks flow-induced changes in gene transcription ( 9 ), suggesting a role for the cytoskeleton in flow-dependent signal transduction. Although several agents that disrupt the cytoskeleton have been shown to affect agonist-induced eNOS activation in endothelial cells ( 22, 47, 48, 52 ), the underlying mechanism is not clear. Given the many influences of the cytoskeleton, several possibilities could account for regulation of eNOS, which, however, remain to be determined.
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( M* M3 _" R: d( J( N" a- ~In endothelial cells, flow-induced eNOS activation is dependent on phosphorylation of eNOS at Ser1179 ( 12 ). Because luminal flow increased eNOS activity and induced its translocation, we tested whether flow would also increase phosphorylated eNOS at the apical membrane. We found that increasing luminal flow caused a similar shift in P-eNOS subcellular localization, with an average increase of 120% at the apical membrane of THALs. Because eNOS phosphorylation at Ser1179 is associated with an increase in enzymatic activity and NO production, our data suggest that eNOS may be phosphorylated before or while it moves to the apical membrane, resulting in increased NO production. Several kinases, such as Akt, protein kinase A, and protein kinase G, have been shown to phosphorylate eNOS at this residue and increase its activity ( 1 ); however, it is not clear which of these protein kinases mediates flow-induced eNOS activation in the THAL.
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" F* l2 F* Z0 M( ^- p* a" FLuminal flow rates in the THAL and along the nephron fluctuate under physiological conditions. Luminal flow rates at the beginning of the rat distal tubule may be as low as 2 nl/min or as high as 34 nl/min during induced diuresis, as measured in free-flow micropuncture experiments ( 7, 19, 23, 24 ). Thus the perfusion rate of 20-25 nl/min we used in our experiments is within the physiological range. Luminal flow rates also oscillate under physiological conditions, with a frequency of 3/min, due to oscillations in the tubuloglomerular feedback (TGF) mechanism ( 20, 21 ). In addition to these small fluctuations in flow rate, contractions of the renal pelvis in rats and hamsters have been shown to constrict the inner medulla, decreasing the luminal size of inner medullary collecting ducts and medullary thin descending and ascending loops of Henle ( 41, 44 - 46 ). These papillary constrictions are likely to halt luminal flow through the THAL, distal tubule, and collecting ducts briefly and in a cyclical manner, significantly altering luminal flow rates (for a review, see Ref. 8). Thus luminal flow rates likely change under physiological conditions; however, it is not known whether these changes are able to induce NO production by the THAL or how this mechanism contributes to overall nephron function.
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In an isolated THAL, increasing luminal flow will likely enhance shear stress, increase transmural pressure across the cells, and also increase mechanical stress on the apical membrane of the cells. In the vasculature, not only shear stress but also changes in vessel wall pressure or stretch have been shown to activate eNOS ( 11, 25 ). Thus any of these stimuli may be responsible for eNOS activation in the THAL. Similar to principal cells in the collecting duct, a primary cilium has been observed in THAL cells ( 3, 26 ). Because most data suggest that the cilium works as a flow sensor in most cells ( 38, 39 ), it is possible to speculate that a primary cilium is involved in flow sensing and eNOS activation in the THAL. However, the precise mechanism that triggers eNOS activation and translocation in the THAL remains to be determined.
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NO exerts potent natriuretic and diuretic effects in the kidney due, in part, to direct inhibition of tubular NaCl and water reabsorption ( 31 ). In the THAL, NO acts in an autocrine manner to inhibit net Cl and bicarbonate absorption; in addition, NO produced by the THAL can act in a paracrine fashion to regulate physiological processes in adjacent cells, such as TGF ( 29, 30, 36, 49 ). Thus regulation of NO production by the THAL is likely to be important to overall regulation of sodium and water balance. While luminal flow has been shown to modulate ion transport in the proximal tubule ( 40 ) and collecting ducts ( 43 ), it is not known whether flow regulates ion transport in the THAL. Our data suggest that increased luminal flow should decrease net NaCl absorption via NO production; however, to our knowledge, this hypothesis has not yet been tested experimentally.
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We conclude that increasing luminal flow stimulates eNOS and induces its translocation to the apical membrane in rat THALs. An intact cytoskeleton is required for flow-induced eNOS activation and translocation in these epithelial cells. eNOS activation by luminal flow may be very important to the regulation of renal medullary NO levels, which may affect tubular and vascular function in the renal medulla.: Q& h7 N( e. I
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GRANTS
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2 b' v( Y. l$ i/ g& IThis work was supported in part by National Heart, Lung, and Blood Institute Grants HL-28982 and HL-70985 (to J. L. Garvin).
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