N ifric Oxide In h Ibits Sod iu m Rea bsorption in the Isolated Perfused Cortical Collecting Duct1
Barbara Anne Stoos, Nestor Horacio Garcia, and Jeffrey Lawrence Garvin2
R ecent studies indicate that nitric oxide (NO) is BA. Stoos. N.H. Garcia, J.L. Garvin, Hypertension and produced by the kidney and plays an important Vascular Research Division, Department of Medicine, role in the regulation of renal sodium excretion. NO Henry Ford Hospital, Detroit. Ml may induce natniuresis by inhibiting sodium reab- sorption by the nephron or by altering hemodynamics. (J. Am. Soc. Nephrol. 1995; 6:89-94) Several in vivo studies support the hypothesis that NO-induced changes in sodium excretion are due to ABSTRACT direct tubular effects. Lahera et at. ( 1 ) reported that Indirect evidence suggests that nitric oxide inhibits NO synthesis blockade with how doses of NG nltro L arginine in rats can reduce sodium excretion, urine sodium reabsorption by the collecting duct; however, flow rate, and filtration fraction without altering mean direct evidence is lacking. It was hypothesized that arterial pressure, RPF, or GFR. In other studies (2,3), endothelium-derived nitric oxide inhibits sodium flux the stimulation of NO release by the kidney induced in the cortical collecting duct by blocking amiloride- natriuresis and diuresis without affecting GFR. There sensitive sodium channels. Tubules were obtained are also in vitro data from our laboratory (4,5) that from Sprague-Dawley rats pretreated with deoxycor- indicate that NO inhibits sodium flux in the nephron. ticosterone acetate (5 mg/rat i.m.) 5 to 9 days before We demonstrated a direct effect of endothehium-de- the experiment. Nitric oxide was added to the system rived nitric oxide (EDNO) on sodium flux in cultured by either the addition of endothelial cells and the cortical collecting duct (CCD) cells. The bradykinin- induction of the release of nitric oxide via acetylcho- stimulated release of EDNO from cultured endothehiah cells decreased short-circuit current (a measure of net line (10 M) or by the addition of nitric oxide donors. active transport) by reducing huminal- to-basolateral Acetylcholine-induced nitric oxide release from en- sodium flux via the inhibition of apical membrane dothelial cells decreased lumen-to-bath sodium flux sodium channels in cultured M- 1 CCD cells. by 24 ± 7% (N = 3; P < 0.05). The addition of the nitric Because cultured cells provide only indirect evi- oxide donor, spermine NONOate (10 M), de- dence for physiologic effects in native cells, the goal of creased net sodium flux 68% from 10.1 ± 2.0 to 3.6 ± this study was to examine the effects of NO on sodium
2 pmol/mm ‘ mm (N = 5; P < 0.025). To assure that the transport in the isolated perfused CCD. Our results inhibition of sodium flux was due to nitric oxide, show that NO released from cultured endothehial cells another donor, nitroglycerin (2 x iO M), was used, or NO donors inhibits sodium reabsorption in the which decreased sodium flux by 43%. Luminal amilo- isolated CCD, as determined by decreases in Intracel- lular sodium and sodium flux, and that this inhibition ride (10 1 M) decreased net sodium flux by 83% (from appears to occur via amiboride-sensitive sodium chan- 14.8 ± 1.2 to 2.4 ± 0.7 pmol/mm ‘ mm; N = 5; P < nebs, rather than via Na -K ATPase. 0.025). The addition of nitric oxide via spermine NONOate to tubules decreased intracellular sodium METHODS levels by 26% (N = 6; P < 0.005). The Na -K ATPase Preparation of the Isolated Nephron Segments activity of spermine NONOate-treated tubules was CCD were obtained from Spnague-Dawley rats ( 120 to 14.7 ± 3.2 pmol/mm . mm compared with the control 1 50 g) that had been pretreated with deoxycorticosterone ± . value of 10.2 2.0 pmol/mm mm. Nitroglycerin did acetate (5 mg/rat i.m.) 5 to 9 days earlier. Deoxycorticoster- not significantly affect pump activity either. It was one acetate treatment was necessary to obtain measurable concluded that: ( 1) nitric oxide inhibits sodium flux in sodium transport. On the day of the experiment, rats were the cortical collecting duct, and (2) this occurs at anesthetized with ketamine ( 100 mg/kg body wt i.p.) and the least in part via amiloride-sensitive sodium channels. abdominal cavity was opened to expose the kidney. The kidney was bathed in ice-cold saline and removed. Coronal slices were cut and placed in physiologic saline at l2 C. CCD Key Words: Nephron transport. amioride, sodium flux. Na - were dissected from medullary rays in the cortex in the same K ATPase solution under a stereomicroscope.
CCD Perfusion
) Received October 25, 1994. Accepted April 3, 1995. The CCD (ranging from 0.45 to 1 .25 mm in length) were 2 Correspondence to Dr. J.L Garvin, Henry Ford Hospital, Hypertension and transferred to a temperature-regulated chamber and per- Vascular Research Division, Detroit, Ml 48202. fused between concentric glass pipettes at 37#{176}Cas described 1046-6673/0601-0089$03.00/0 Journal of the American society of Nephrology previously (6). The composition of the basohateral bath and copyright C 1995 by the American society of Nephrology penfusate, in millimolar concentrations, was: NaC1, 1 14;
Journal of the American Society of Nephrology 89 NO Inhibits Na Flux in COD
NaHCO3. 25; NaH2PO4, 2.5; KC1, 4; MgSO4, 1 .2; alanine, 6; 10 to 20 mm, six more collections of penfusate were made Na3citrate, 1 : glucose, 5.5; Ca-lactate2, 2; rafllnose, 5. The and sodium concentrations were determined as before. solution was bubbled with 5% CO2 and 95% 02 before and during the experiment. The basohateral bath was exchanged Intracellular Sodium Measurements at a rate of 0.5 mL/mln, and tubules were perfused at Once the CCD were perfused, cells were loaded at 37#{176}C approximately 2 nL/mm ‘ min. In preliminary experiments, with the fluorescent dye SBFI-AM (Molecular Probes, Eu- it was determined that tubules did not absorb significant gene, OR) by the addition of 5 prnoh of dye and 0.05% quantities of fluid. phuronic acid in dimethylsulfoxide to the bath for 60 mm. At the end of the incubation period, dye and pluronic acid were Unidirectional Sodium Flux washed from the bath and the tubules were incubated for an Tubules were perfused as described above, and then, 22Na additional 30 mm before the experiment was begun. Fhuores- was added to the perfusate (25 pCi/mL). Twenty minutes cence was imaged digitally with an Image intensifier (Video was allowed for isotopic equilibration. At the end of the Scope International, Herndon, VA) and a CCD camera 20-mm incubation period. three collections of bath solution (Hamamatsu, Hamamatsu City, Japan). Digital images of an were made during the control period. After the control period, area where the tubule contained only principal cells were 200 L ofendothehial cells (cow pulmonary artery endothehial analyzed with an Image One MetaFluor system (Universal cells: America Type Culture Collection CCL 209, Rockville, Imaging. West Chester. PA). Intracellular sodium was deter- MD: cultured on microcarnier beads) was added to the bath mined from the ratio of fluorescence at excitations of 340 and (volume, 1 mL) and the tubule was positioned on top of the 380 am. At the end of the wash period, measurements were endothehial cells. Acetylcholine (Ach. iO M) was then added taken once a minute for 5 min, after which 10 moh of to the bath to stimulate the release ofEDNO. Twenty minutes spermine NONOate was added to the bath and ratios were later, three collections of bath solution were made. Unidirec- monitored once a minute for an additional 25 min. Fluores- tional lumen-to-bath sodium flux was calculated from the cent ratios were calibrated in situ by permeabilizing cells with appearance of the isotope in the bath. (Endothebial cells nystatin (334 U/mL) and altering the sodium concentration could not be added at the beginning of the control period of the bath and perfusion solution (Figure 1). because the shear stress associated with transferring them to the chamber and/or the flowing bath stimulated them to Na -K ATPase Assay release EDNO.) ATPase activities of control CCD and COD treated with NO Once we had demonstrated that EDNO released from donors were measured in the presence and absence of endothehial cells inhibited lumen-to-bath sodium flux. we ouabain according to the methods of O’Neil and Dubinsky abandoned this protocol because: ( 1 ) the success rate was (8). CCD were incubated in perfusate solution for 20 min at very low: while adjusting the position of the endothebial cells 37#{176}Cwith either vehicle, spermine NONOate (10 M), or so that the collection pipet could be seen, we often knocked nitroglycerin (2 x iO M). In order to obtain total and the tubule off the perfusion pipettes and (2) the concentra- ouabain-insensitive ATPase activity, the tubules were trans- tion of NO was unknown. ferred to 40 L of ATPase incubation solution containing (in
Net Sodium Flux 2.2 The effects of two NO donors: ( 1 ) spermine NONOate (1,3 propanediamine. N-14-[ 1 -(3-aminopropyh)-2-hydroxy-2-ni- 0 trosohydrazinol-butyhIC10H26N6O2: Cayman Chemical Co. Co c ) 2.0 Ann Arbor, MI), and (2) nitroglycerin (2 X iO M: DuPont 0 Pharmaceuticals, Manati, Puerto Rico) were tested on net , i. sodium flux in isolated CCD. After six control measurements, Cl) 0 an NO donor was then added to the bath. Twenty minutes I .8 later, six additional collections were made. Sodium concen- ck . trations of samples of perfusate and collected fluid were measured In an instrument with a sodium-sensitive glass C.) 1.6 detector (7). C) r = 0.998 Because sodium reabsorption was not accompanied by 0 Cl) significant fluid reabsorption, net sodium flux (JNa) was C) 01 calculated according to I .4
U- ‘JNa PR([N l - lNaLI)
where PR is the perfusion rate normalized for tubule length. 1.2 I I I Na is the sodium concentration In the perfusion solution, 0 10 20 30 40 50 60 and NaL is the sodium concentration collected from the Sodium Concentration (mM) tubule.
Figure 1. Representative calibration curve for SBFI depicting Amiloride-Sensitive Sodium Flux sodium concentration versus ratio of fluorescence intensity
Net Na was also measured In the presence of amilonide (10 at 340 and 380 nm. Calibration was performed in situ at the jIM; Sigma, St. Louis, MO). Amiloride was prepared on the end of the experiment. Collecting ducts were loaded for day of the experiment and added to the perfusate after approximately 60 mm with 5 pM SBFI. Cells were permeabi- collections had been made from the tubule during the control lized with nystatin, and the sodium concentration in the bath period. After the tubule had been exposed to the amilonide for and perfusate changed as Indicated in the figure.
90 Volume 6 ‘ Number 1 1995 Stoos et al
milhimolar concentrations): NaCl, 120: KC1, 30; imidazole, Because adding endothebial cells to the bath often 50: MgCh2. 5: EGTA, 0.5; Na2ATP, 5; phosphoenol pyruvate. damaged the tubule and the concentration of NO to 10; ascorbic acid, 1 : NADH, 1 , plus 1 .4 U/mL pyruvate which CCD were exposed was difficult to control in the kinase and 2.0 U/mL lactate dehydrogenase: either in the previous experiments, we switched to NO donors. absence or presence of ouabain (4 X i0 mM). After the First, we examined the effects ofspermine NONOate, a tubules had been placed In the appropriate solution. they donor that spontaneously releases NO. In these and were incubated for 30 min at 37#{176}C.The reaction was stopped by adding 20 .tL of 1 .0 N HC1 and maintained at room subsequent experiments, we measured net sodium temperature for 1 5 mm. A total of 50 j L of the acidified reabsorption ( Na)#{149} The addition of spermine NONOate mixture was added to 750 L of 6 N NaOH and then placed in ( i0 M) resulted in a 68.4 ± 15.5% decrease in Na’ a sand bath for 20 mm at 60#{176}Cinthe dark to convert NAB to from 10. 1 ± 2.0 to 3.6 ± 2.0 pmoh/mm . mm (N = 5: P a condensed form of NAD. The tubes were allowed to cool to < 0.025; Figure 3A). The addition of the vehicle for room temperature in the dark. Fluorescence was read at 340 spermine NONOate produced no significant change in nm of excitation and 460 nm of emission. The difference Na (from 15.4 ± 2.0 to 17.6 ± 3.0 pmol/mm . mm; N between total and ouabain-insensitive ATPase activity was = 7). The difference In the inhibition of sodium reab- taken to be Na -K ATPase activity. sorption by NO rebeased from endothehial cells versus spermine NONOate may reflect differences in NO con- Statistics centration in the basolateral bath. Experimental results are expressed as mean ± SE. Data To assure that the Inhibition ofsodium reabsorption were evaluated with a paired t test. The criterion for statisti- caused by endothebial cells and spermine NONOate cal significance was P < 0.05. was the result of NO, we used another NO donor, RESULTS nitroglycerin. During the control period. sodium reab- sorption was 10.0 ± 1 .2 pmob/mm . mm; after i0 M Because EDNO has been reported to be natriuretic, nitroglycerin was added to the bath, it decreased to we examined its effects on sodium reabsorption by 5.4 ± 1 .4 pmoh/mm ‘ mm, an inhibition of 43. 1 ± isolated, perfused CCD. Initially, we used cultured endothelial cells as a source of EDNO and measured unidirectional lumen-to-bath sodium flux with 22Na. A During the control period, sodium reabsorption was 20 z 52.7 ± 4.0 pmol/mm . mm. After endothelial cells and 0 Ach ( i0 M) were added to the bath, flux decreased Q by 24.0 ± 6.6% (N = 3; P < 0.05; Figure 2). In the 0. U, absence of endothehial cells, Ach had no significant mE < E io effect on unidirectional sodium flux (Figure 2). These data suggest that the Ach-induced release of NO from 00 endotheblal cells inhibits lumen-to-bath sodium reab- OE sorption. ti z 0 10 Ach CONTROL SPERUINE plus NONOA1t a) B endothelial cells Ach 20 0 - z 0 C) F:-.. 0 . 15 I 0. -10 U) U- 10 ,.-. z -20 OE 5
0 0 z a) -30 0 CONTROl NrTROGLYCERIN
Figure 3. (A) Spermine NONOate inhibits Na in perfused -40 CCD. The addition of spermine NONOate to the basolateral Figure 2. Ach-induced release of NO from endothelial cells bath resulted In a 68.4 ± 5.5% decrease in Na’ from 10.1 ±
inhibits the unidirectIonal sodium flux in perfused CCD. The 2.0 to 3.6 ± 2.0 pmol/mm . mm (N = 5; P < 0.025). (B) addition of iO M Ach in the absence of endothelial cells Nitroglycerin inhibits Na In perfused CCD. The addition of had no significant effect on sodium flux. The addition of Ach nitroglycerin to the basolateral bath resulted In a 43. 1 ±
in the presence of endothelial cells decreased sodium flux 16.7% decrease in Na’ from 10.0 ± 1.2 to 5.4 ± 1.4 pmol/
by24 ± 6.6%(N= 3; P< 0.05). mm mm (N = 7; P < 0.05).
Journal of the American Society of Nephrology 91 NO Inhibits Na Flux in COD
16.7% (N = 7: P < 0.05; Figure 3B). The addition of the In a second set of experiments, we examined the vehicle produced no significant change in Na (from effect of nitroglycerin. Total ATPase activity in control
15 ± 1 .7 to 13.8 ± 1 . 1 pmol/mm . mm: N = 5). These tubules was 19.8 ± 4.2 pmol/mm . mm, and ouabain- experiments support the hypothesis that NO directly insensitive ATPase activity was 10.7 ± 3.8 pmol/ inhibits sodium reabsorption by the CCD. mm ‘ mm, giving an Na -K ATPase activity of 9. 1 ±
To determine whether NO-induced changes in so- 4.0 pmol/mm ‘ mm. In nitroglycerin-treated (2 X iO dium flux were due to alterations in sodium entry or M) tubules, total ATPase activity was 23. 1 ± 2.6 exit, we measured Na -K ATPase activity and intra- pmoh/mm . mm and ouabain-insensitive ATPase was cellular sodium concentration. First, we investigated 10.2 ± 2.2 pmob/mm ‘ mm; thus, Na -K ATPase ac- whether NO affects Na -K ATPase activity in isolated tivity was 1 2.9 ± 2.6 pmob/ mm mm, not significantly
COD under maximum rate conditions. In the first set different from the controls (N = 5; Figure 4B) or from of experiments, we examined the effect of spermine the experiments with spermine NONOate. These ex- NONOate. Total ATPase activity in control tubules periments suggest that the inhibition of Na induced was 20.9 ± 1 .2 pmol/mm . mm. Ouabain-insensitive by NO donors is not due to the suppression of the ATPase activity was 10.8 ± 1 .4 pmol/mm . mm; con- Na -K ATPase. sequently. Na -K ATPase activity was 10.2 ± 2.0 To assure that EDNO was affecting sodium entry pmoh/mm . mm. In spermine NONOate-treated ( i05 rather than sodium exit, we measured the effect of M) tubules, total ATPase activity was 22.2 ± 1.8 spermine NONOate on intracellular sodium concen- pmol/mm . mm and ouabain-insensitive ATPase was tration (Figure 5). During the control period, intracel- 7.5 ± 1 .7 pmoh/mm . mm; thus, Na -K ATPase activ- lular sodium was 2 1 .3 ± 6.8 mM. Spermine NONOate ity was 14.7 ± 3.2 pmoh/mm . mm, not significantly (10 M) caused it to fall by 5.6 ± 1.1 mM to 15.8 ± different from the controls (N = 6; Figure 4A). 7.3 mM (N = 6; P < 0.005). These experiments support the hypothesis that NO Inhibits sodium reabsorption by affecting sodium entry into the cell rather than exit. A Two means of sodium entry across the apical mem- brane of the COD have been described, an amiloride- = CONTROL sensitive sodium channel and a thiazide-sensitive, - SPERUINE NONOATE (1 05M)
,,- C electroneutral transporter. In order to determine EE whether NO inhibits sodium reabsorption via amilo- I- ride-sensitive sodium channels or another pathway, 0 Na was measured in the presence of amionide. Dur- V ing the control period, Na was 14.8 ± 1 .0 pmoh/ a.! mm mm. The addition of amioride ( 10 j.tM) to the nini
B 30 = CONTROL a) -.. 25 1 NITROGLYCERIN (2 X 10514) -#{176}- C z30- I- . 20 oE a) 4: E C) 20 0 a)
C 10 u1 rhi I TOTAL OUABMN- Na+/K+ INSENSIITVE 0 Figure 4. (A) The effect of spermine NONOate on ATPase activity in isolated CCDs. There was no significant difference control spermine in total, ouabaln-insensitive, or Na -K ATPase activity of CCD NONOate between spermine NONOate-treated and control tubules (N
= 6; not significant). (B) Effect of nitroglycerin on ATPase Figure 5. NO reduces intracellular sodium concentration in activity in isolated CCD. There was no significant difference CCD. Tubules were treated with NO by the addition of in total, ouabain-insensitive, or Na -K ATPase activity of CCD spermine NONOate (10 M) to the bath. Spermine NONO- between nitroglycerln-treated and control tubules (N = 5; ate reduced intracellular sodium concentration by 26% (N = not significant). 6; P< 0.005).
92 Volume 6 ‘ Number 1 . 1995 Stoos et al
perfusate decreased Na 11ux by 83. 1 ± 3.2%, from tion in the CCD and may explain the natriuretic effect 14.8 ± 1.0 to 2.4 ± 0.4 pmol/mm mm (N = 5; P < seen in whole-animal studies. 0.025; Figure 6). demonstrating that sodium reab- Sodium reabsorption by the CCD can be described sorption In these tubules is accomplished mainly as a two-step process: entry via a channel or electro- through electrogenic amiloride-sensitive channels. neutral transporter and exit via Na -K ATPase. The inhibition of sodium flux could be a result of either
reduced entry or exit. The measurement of Na - DISCUSSION K ATPase activity in the tubules under maximum rate This is the first study, to our knowledge, to demon- conditions showed no significant difference in the strate that NO derived from either endothelial cells or presence or absence of NO. These data suggest that chemical donors directly Inhibits sodium reabsorption NO does not reduce pump activity. It is possible that in the COD of the rat. NO inhibits Na at least In part NO inhibits sodium reabsorption by increasing the K’ by blocking amiloride-sensitive sodium channels, be- for sodium or potassium; however, this seems un- cause (1 ) amiloride inhibited 83% of Na; (2) NO likely, because the measurement of Na -K ATPase blocked Na up to 68%; and (3) NO had no significant activity with sodium and potassium concentrations effect on Na /K ATPase activity. that approximate the K#{189}for each ion in cultured CCD These results support in vivo data that suggest that (5) revealed no difference when measured in the ab- NO exerts direct effects on urinary sodium excretion sence and presence of NO. ( 1 .9-1 1). It has been demonstrated that when NO To assure that NO was affecting entry, we also synthesis inhibitors are administered intrarenally, measured Intracellular sodium concentration. If NO they lower sodium excretion (9-1 1) and NO donors blocks the entry process, intracellular sodium would induce natriuresis ( 1 2), whereas agents (bradykinin or be expected to fall. Intracellular sodium levels In the Ach) that stimulate NO synthesis cause natriuresis isolated COD decreased 26% after NO was added. This (2,3, 13) without affecting GFR. Thus, these data mdi- likewise suggests that NO inhibits the apical mem- cate that NO exerts a tubular effect to regulate sodium brane and not the basolateral Na -K ATPase, be- excretion. Our data indicate that at least part of the cause lithe pump were inhibited, Intracellular sodium tubular effect resides in the collecting duct where NO measurements would initially increase rather than Inhibits sodium reabsorption. decrease. To determine the effects of NO on Na CCD, NO Our data from the isolated, perfused COD showing was added to the system, either by the addition of that NO inhibits sodium entry are similar to our endothellal cells and the induction of the release of NO previous finding that NO reduces active sodium reab- via Ach or by the addition of NO donors to the baso- sorption by cultured cells (5). These studies showed lateral bath. We found that NO released from both that the addition of nystatin. a cation-selective iono- sources inhibited sodium reabsorption. As with EDNO phore. reversed the effect of EDNO on short-circuit released from endothellal cells, NO released from do- current and that NO had no effect on the maximum nors Inhibited sodium reabsorption. The larger Inhi- rate of pump turnover or the affinities for sodium and bition of Na induced by the NO donors. spermine potassium.
NONOate (68%), and nitroglycerin (43%), compared Na the COD has been reported to occur via with that which occurred in stimulated endothelial amiloride-sensitive sodium channels ( 14-16) and an cells, may reflect differences in the NO concentration electroneutral process ( 1 7, 18). In our studies, amilo- In the bath. Taken together, these data support the ride inhibited 83% of Na’ Indicating that Na occurs hypothesis that NO directly inhibits sodium reabsorp- primarily via sodium channels. We found that NO
decreased Na by as much as 68%; thus, NO must decrease Na at least in part by blocking sodium 20 channels. z 0 This appears to be the first work demonstrating the direct effect of the NO-induced Inhibition of sodium transport in the isolated, perfused collecting duct. 0 4,... U, Others have shown that NO may inhibit sodium reab- Eio sorption in the proximal tubule ( 19.20). Guzman et at. ( 1 9) treated mouse proximal tubule epithelial cells OE with lipopolysaccharide ( 10 g/mL) and interferon ( 100 U/mL) to induce NO synthase, resulting in max- z imal inhibition of Na -K ATPase activity at 24 h. The levels of NO in those experiments were not measured; CONTROL AUILORIDE however, because the inducible form of the enzyme
Figure 6. Amiloride inhibits Na in perfused CCD. The addition was stimulated, a high concentration of NO is proba- of amiloride (10 M) to the perfusate reduced JNa by 83. 1 ± ble and may account for the results. Roczniak and
3.2%, from 14.8 ± 1 .0 to 2.4 ± 0.4 pmol/mm . mm (N = 5; P < Burns (20) demonstrated that sodium nitroprusside 0.025). caused the concentration-dependent inhibition of
Journal of the American Society of Nephrology 93 NO Inhibits Na Flux in COD
Na -H F exchange and increased cGMP production in Endothelium-derived relaxing factor inhibits transport proximal tubules. Maximal inhibition of NatH ex- and increases cGMP content in cultured mouse cortical collecting duct cells. J Clin Invest 1992:89:761-765. change (38%) occurred, but at a concentration of 10 5. Stoos BA, Carretero OA, Garvin JL: Endothelial-derived M, two orders of magnitude higher than in this study. nitric oxide inhibits sodium transport by affecting apical Furthermore, those investigators did not examine any membrane channels in cultured collecting duct cells. J Am Soc Nephrol 1994;4:1885-1860. other NO donor to assure that these effects were 6. Garvin JL, Burg MB, Knepper MA: Ammonium replaces indeed due to the release of NO and not specific to the potassium in supporting sodium transport by the Na-K drug itself; therefore, the physiologic relevance of this ATPase of renal proximal straight tubules. Am J Physiol 1985:249:F785-F788. finding is not clear. 7. Garvin JL: A simple method to determine millimolar The origin of NO in vivo is speculative at this time; concentrations of sodium in nanoliter samples. Kidney however, it appears quite plausible that NO can dif- Int 1993;44:875-880. fuse from endothelial cells in the renal cortex to the 8. O’Neil RG, Dubinsky WP: Micromethodology for measur- Ing ATPase activity in renal tubules: mineralocorticoid collecting duct (4), where it may induce natriuresis. A influence. Am J Physiol 1984:247:C314-C320. similar hypothesis was originally put forth by Majid et 9. Majid DSA, Navar LG: Suppression of blood flow auto- at. ( 1 0). who proposed that increases in renal shear regulation plateau during nitric oxide blockade in canine kidney. Am J Physiol 1992:31:F40-F46. stress enhanced NO release and, consequently, natri- 10. Majid DSA, Williams A, Navar LG: Inhibition of nitric uresis. Another possibility is that the collecting duct oxide synthesis attenuates the pressure induced natri- itself produces NO, thereby exerting an autocrine uretic response in anesthetized dogs. Am J Physiol 1992; 264:F79-F87. effect on sodium reabsorption. Terada et at. (2 1 ) dem- 1 1 . Salom MG, Lahera V. Miranda-Guardiola F, Romero JC: onstrated by polymerase chain reaction coupled to Blockade ofpressure natriuresis induced by inhibition of reverse transcription that the message for the consti- renal synthesis of nitric oxide in dogs. Am J Physiol 1992:262:F7 18-F722. tutive form of NO synthase is present in both the CCD 12. Majid DSA, Williams A, Kadowitz PJ. Navar LG: Renal and the inner medullary collecting duct. Furthermore, responses to intra-arterial administration of nitric oxide Mohaupt et a!. (22) recently reported that both CCD donors in dogs. Hypertension 1993;22:535-541. 13. Baer PG. Navar LG, Guyton AG: Renal autoregulation. and inner medullary collecting ducts also express an ifitration rate, and electrolyte excretion during vasodila- inducible form of NO synthase. This suggests that NO tion. Am J Physiol 1970;219:619-625. may not need to diffuse from the vasculature at all but 14. Rouch AJ, Chen L, Troutman SL, Schafer JA: Na may be produced within the nephron. transport in isolated rat CCD: effects ofbradykinin, ANP, clonidine, and hydrochlorothiazide. Am J Physiol 1991; In summary, we have found that NO inhibits so- 260:F86-F95. dium reabsorption by the CCD. This inhibition is most 15. Stokes JB: K secretion by cortical collecting tubule: likely due to the blockade of sodium entry via apical relation to Na absorption, luminal Na concentration, and transepithelial voltage. Am J Physiol 198 1 :24 1 :F395- membrane sodium channels, because NO reduced F402. intracellular sodium concentration but did not alter 16. Re1IMC, Troutman SL, SchaferJA: Sodium transport by Na / K ATPase activity and sodium reabsorption was rat cortical collecting tubule. Effects of vasopressin and deoxycorticosterone. J Chin Invest 1986:77: 129 1-1298. found to occur primarily via amiloride-sensitive so- 1 7. Tomita K, Pisano JJ, Knepper MA: Control of sodium dium channels. and potassium transport in the cortical collecting duct of the rat. Effects ofbradykinin, vasopressin and deoxycor- ACKNOWLEDGMENTS ticosterone. J Chin Invest 1985:76:132-136. 18. Terada Y, Knepper MA: Thiazide-sensitive NaCl absorp- This work was supported in part by Grant HL 28982 from the NIH and tion in rat cortical collecting duct. Am J Physiol 1990; a Research Career Development Award (HL 0289 1) to J.L. Garvin. 259:F5 19-F528. 19. Guzman NJ, Fang MZ, Tang 55, IngelfingerJR, Garg LC: REFERENCES Autocrine inhibition of proximal tubule Na /K ATPase by nitric oxide [Abstractj. Hypertension 1994;24:380. 1 . Lahera V. Salom MG, Miranda-Guardiola F, Moncada S. 20. Roczniak A, Burns KD: Exogenous nitrates increase Romero JC: Effects of NG nitro L arginine methyl ester cyclic GMP (cGMP) and inhibit Na -H exchange in on renal function and blood pressure. Am J Physiol rabbit proximal tubule (PT) [Abstracti. J Am Soc Nephrol l991;261:F1033-F1037. 1994:5:259. 2. Lahera V. Salorn MG, Fiksen-Olsen MF, Raij L, Romero 2 1 . Terada Y, Tomita K, Nonoguchi H, Marumo F: Poly- JC: Effects of N ’-monomethyl-L-arginine and L-arginine merase chain reaction localization of constitutive nitric on acetylcholine renal response. Hypertension 1990; 15: oxide synthase and soluble guanylate cyclase messenger 659-663. RNAs in microdissected rat nephron segments. J Clin 3. Lahera V, Salom MG, Fiksen-Olsen MF, Romero JC: Invest 1992:90:659-665. Mediatory role of endothelium-derived nitric oxide in 22. Mohaupt MG, Elzie JL. Ahn KY, Clapp WL, Wilcox CS, renal vasodilatory and excretory effects of bradykinin. Kone BC: Differential expression and induction of Am J Hypertens 199 1:4:260-262. mRNAs encoding two inducible nitric oxide synthases in 4. Stoos BA, Carretero OA, Farhy RD. Scicli G, Garvin JL: rat kidney. Kidney Int 1994;46:653-665.
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