Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our laboratory has previously shown that
mice lacking neuronal nitric oxide synthase (nNOS) are defective in
fluid absorption (Jv) and HCO1 · mm
1,
n = 13, P < 0.01) and
Jv was 38% lower (0.95 ± 0.15 vs.
1.54 ± 0.17 nl · min
1 · mm
1,
n = 13, P < 0.05) in iNOS knockout mice
compared with their wild-type controls. Addition of the iNOS-selective
inhibitor L-N6-(1-iminoethyl)
lysine, reduced both Jv and
JHCO3 significantly in wild-type, but not in
iNOS knockout, mice. In contrast, both JHCO3
(93.3 ± 7.9 vs. 110.6 ± 6.18 pmol · min
1 · mm
1) and
Jv (1.56 ± 0.17 vs. 1.55 ± 0.16 nl · min
1 · mm
1) did not
change significantly in eNOS knockout mice. These results indicated
that iNOS upregulates Na+ and HCO
nitric oxide synthase; inducible nitric oxide synthase; endothelial nitric oxide synthase; neuronal nitric oxide synthase; knockout mice; kidney tubule; transport
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THREE ISOFORMS OF NITRIC OXIDE (NO) synthase (NOS), neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), have been identified (20, 34). Immunocytochemical studies have shown that all three isoforms of NOS are expressed in the kidney. eNOS is expressed in renal vascular endothelial cells (26, 34), and nNOS has been found predominantly in epithelial cells of the macula densa (22, 26), as well as in principal cells of the collecting duct (33). iNOS is widely expressed in tubule epithelia, including the proximal tubule, thick ascending limb, and distal convoluted tubule (21, 34). Renal proximal tubules and inner medullary collecting duct cells can produce NO by means of expression of iNOS (1). However, the role of iNOS and eNOS in proximal tubule transport is not clear.
Recently, this laboratory showed that both HCO
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animal preparation and surgical procedures. iNOS and eNOS knockout and wild-type control mice were obtained from Jackson Laboratories, and the ages and sexes were matched between knockout and control mice. They were maintained on a regular diet and tap water until the day of the experiment. The mice were anesthetized by intraperitoneal injection of 100 mg/kg body wt of Inactin [5-ethyl-5-(L-methylpropyl)-2-thiobarbituric acid; Byk-Gulden, Konstanz, Germany] and placed on a thermostatically controlled surgical table to maintain body temperature at 37°C. After a tracheotomy, the left jugular vein was exposed and cannulated with a PE-10 catheter for intravenous infusion. A carotid artery was also catheterized with PE-10 tubing for collection of arterial blood for blood-gas analysis and measurement of mean arterial pressure. Blood-gas analysis was performed on freshly drawn blood with a Corning Blood Gas Analyzer.
Microperfusion of proximal tubule in vivo. Superficial proximal tubules were perfused in vivo by a method similar to those described previously (27, 32). On completion of surgery, 0.3% body wt of isotonic saline was given intravenously to replace surgical fluid losses. Saline solution (0.9%) was continuously infused at a rate of 0.15 ml/h. The left kidney was exposed through a lateral abdominal incision, carefully isolated, and immobilized in a kidney cup filled with light mineral oil (37°C). The kidney surface was illuminated by a fiber optic light. A proximal convoluted tubule with three to five loops on the kidney surface was selected and perfused with a Hampel-type microperfusion pump at a rate of 15 nl/min with a proximal oil block. Tubule fluid collections were made downstream by using another micropipette with oil block on the distal side. The perfusion solution contained 20 µCi/ml of low-Na+ [3H]methoxyinulin for measuring volume absorption and 0.1% FD&C green for identification of the perfused loops. After collection of perfusion fluids, the perfused tubules were marked with heavy mineral oil stained with Sudan black.
The composition of the perfusion fluid was (in mM) 115 NaCl, 25 NaHCOMeasurement of rate of Jv and JHCO3. The rates of net Jv and JHCO3 were calculated on the basis of changes in the concentrations of [3H]inulin and total CO2 as described previously (6). The concentration of radioactive [3H]methoxyinulin contained within each sample was determined by liquid scintillation spectroscopy. The rate of net Jv was calculated from the changes in [3H]inulin concentration, and the total CO2 concentrations in both initial and collected fluids were measured by the microcalorimetric (picapnotherm) method (29, 30). Jv and JHCO3 are expressed per minute per millimeter of proximal tubule.
Statistics. Data are presented as means ± SE. Two-way Student's t-test was used to compare control and experimental groups. ANOVA and Dunnett's test were used for comparison of several experimental groups with a control group. The difference between the mean values of an experimental group and a control group was considered significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proximal tubule Jv and JHCO3 in iNOS and
eNOS knockout mice.
To focus on the specific role of iNOS and eNOS on proximal tubule
Na+ and HCO1 · mm
1
(n = 13, P < 0.01).
Jv was 0.95 ± 0.15 vs. 1.54 ± 0.17 nl · min
1 · mm
1
(n = 13, P < 0.05). The reduced rates
of JHCO3 in the proximal tubule are of interest
in view of our laboratory's previous demonstration that lack of iNOS
leads to an increase in urine HCO
|
|
Effects of iNOS inhibitor on proximal tubule transport.
To examine whether acute blocking of iNOS activity alters the proximal
tubule Jv and JHCO3, the
effect of a selective iNOS inhibitor, L-NIL, was studied.
L-NIL was given by intravenous injection (1.5 mg/kg) and
was also added to the tubular perfusate (100 µM). As shown in Fig.
2, addition of L-NIL
significantly decreased both Jv and
JHCO3 in wild-type mice;
Jv was 1.54 ± 0.17 and 1.03 ± 0.15 nl · min1 · mm
1
(n = 12, P < 0.05) and
JHCO3 was 67.4 ± 11.9 and 109.9 ± 7.3 pmol · min
1 · mm
(n = 12, P < 0.05) in the absence and
presence of L-NIL, respectively. In contrast,
L-NIL had no effect on either Jv or
JHCO3 in iNOS knockout mice;
Jv was 0.95 ± 0.15 and 0.96 ± 0.27 nl · min
1 · mm
1 and
JHCO3 was 71.7 ± 6.4 and 83.5 ± 6.03 pmol · min
1 · mm
(n = 10, P > 0.05) in the absence and
presence of this inhibitor, respectively.
|
Acid-base status in iNOS and eNOS knockout mice.
To examine the role of iNOS or eNOS in maintaining acid-base balance,
we studied arterial blood pH and HCO
|
Blood pressure and plasma electrolytes in iNOS and eNOS knockout
mice.
To address whether iNOS or eNOS is involved in maintaining normal blood
pressure and plasma electrolyte balance, we examined the plasma
Na+ and K+ concentrations and hematocrit in
iNOS and eNOS knockout mice. The plasma Na+ and hematocrit
values are within normal ranges in wild-type and NOS knockout mice
(Table 2). This finding suggests that
reduced Na+ absorption in proximal tubules was fully
compensated in iNOS knockout mice. These data show the plasma
K+ was higher in eNOS knockout mice than in control
animals; however, the mechanism for this increment is not clear.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our laboratory previously demonstrated that inhibition of NO
synthesis with
NG-monomethyl-L-arginine induces
significant diuresis and natriuresis in both rats and mice (27,
31); blocking basal NO synthesis decreases
Jv and JHCO3 in the
proximal convoluted tubule of both rat and mouse kidney (27,
31). Our laboratory also demonstrated that the nNOS knockout
mouse has hypotension and metabolic acidosis and that proximal tubule
Na+ and HCO
Experimental evidence demonstrates that iNOS is present in proximal tubules (1, 21) and that proximal tubules can produce NO by expression of iNOS (1). This raises the possibility that iNOS may modulate proximal tubule functions. Previous studies suggested that iNOS is important in response to hypoxic injury, because proximal tubules isolated from iNOS null mice (but not from nNOS or eNOS null mice) are resistant to hypoxic injury (19). It has also been shown that the absence of iNOS activity does not protect mice from septic shock after lipopolysaccharide administration (18), consistent with the conclusion that iNOS plays an important role in immune defense (19, 20). The observation that both Jv and JHCO3 are lower in iNOS knockout mice suggests a new function of iNOS in regulation of proximal tubule transport, indicating that under basal conditions iNOS upregulates Na+ absorption and JHCO3 in proximal tubules. Mice lacking iNOS were indistinguishable from wild-type mice in appearance and histology; there was no difference in blood pressure or heart rate (11). This suggests that a compensatory mechanism of upregulation of Na+ absorption and JHCO3 occurs because the proximal tubule Na+ absorption and JHCO3 is lower, but the blood pressure and acid-base balance did not change significantly. This compensation may include both intrarenal and extrarenal mechanisms in regulation of acid-base balance.
The present study, as well as our laboratory's previous data, shows
that urine HCO
Several factors may alter the rate of proximal tubule Na+
and HCO
The physiological importance of the upregulation of proximal tubule transport by iNOS is not clear. It has been reported that increased blood flow rate stimulates NO release and produces vasodilation in segments of rat aorta and in endothelial cells on microcarrier beads. This response is most likely mediated by eNOS (23). Whether increased tubular perfusion rates stimulate NO release by means of iNOS, which then increases transport in the proximal tubule, is not known. Flow-dependent changes in Jv and JHCO3 in the proximal tubule are well documented (3, 24).
Previous studies disclosed two distinct effects of NO actions on
Na+ and HCO
![]() |
ACKNOWLEDGEMENTS |
---|
I thank Drs. R. Berliner and G. Giebisch for reviewing the manuscript, William Sessa for providing constructive comments, and Leah Sanders for assistance in preparation.
![]() |
FOOTNOTES |
---|
Portions of the study were previously published in abstract form (28). This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-17433.
Address for reprint requests and other correspondence: T. Wang, Dept. of Cellular and Molecular Physiology, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06520 (E-mail: tong.wang{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 29, 2002;10.1152/ajprenal.00243.2001
Received 1 August 2001; accepted in final form 17 May 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahn, KY,
Mohaupt MG,
Madsen KM,
and
Kone BC.
In situ hybridization localization of mRNA encoding inducible nitric oxide synthase in rat kidney.
Am J Physiol
36:
F748-F757,
1994.
2.
Alpern, RJ,
Cogan MG,
and
Rector FC, Jr.
Effect of luminal bicarbonate concentration on proximal acidification in the rat.
Am J Physiol Renal Fluid Electrolyte Physiol
243:
F53-F59,
1982
3.
Alpern, RJ,
Cogan MG,
and
Rector FC, Jr.
Flow dependence of proximal tubular bicarbonate absorption.
Am J Physiol Renal Fluid Electrolyte Physiol
245:
F478-F484,
1983
4.
Amorena, C,
and
Castro AF.
Control of proximal tubule acidification by the endothelium of the peritubular capillaries.
Am J Physiol Regul Integr Comp Physiol
272:
R691-R694,
1997
5.
Chan, YL,
Biagi BA,
and
Giebisch G.
Control mechanisms of bicarbonate transport across the rat proximal convoluted tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
242:
F532-F543,
1982
6.
Chan, YL,
and
Giebisch G.
Relationship between sodium and bicarbonate transport in the rat proximal convoluted tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
240:
F222-F230,
1981
7.
Chan, YL,
Malnic G,
and
Giebisch G.
Passive driving forces of proximal tubular fluid and bicarbonate transport: gradient dependence of H+ secretion.
Am J Physiol Renal Fluid Electrolyte Physiol
245:
F622-F633,
1983
8.
Chatterjee, PK,
Patel NS,
Kvale EO,
Cuzzocrea S,
Brown PA,
Stewart KN,
Mota-Filipe H,
and
Thiemermann C.
Inhibition of inducible nitric oxide synthase reduces renal ischemia/reperfusion injury.
Kidney Int
61:
862-867,
2002[ISI][Medline].
9.
De Nicola, L,
Blantz RC,
and
Gabbai FB.
Nitric oxide and angiotension II. Glomerular and tubular interaction in the rat.
J Clin Invest
89:
1248-1256,
1992[ISI][Medline].
10.
Green, M,
Ruiz OS,
Kear F,
and
Arruda JA.
Dual effect of cyclic GMP on renal brush border Na-H antiporter.
Proc Soc Exp Biol Med
198:
846-851,
1991[Abstract].
11.
Huang, PL,
and
Fishman MC.
Genetic analysis of nitric oxide synthase isoforms; targeted mutation in mice.
J Mol Med
74:
415-421,
1996[ISI][Medline].
12.
Huang, PL,
Huang Z,
Mashimo H,
Bloch KD,
Moskowitz MA,
Bevan JA,
and
Fishman MC.
Hypertension in mice lacking the gene for endothelial nitric oxide synthase.
Nature
377:
239-242,
1995[ISI][Medline].
13.
Jaramillo-Juarez, F,
Aires MM,
and
Malnic G.
Urinary and proximal tubule acidification during reduction of renal blood flow in the rat.
J Physiol
421:
475-483,
1990[Abstract].
14.
Kinoshita, Y,
and
Knox FG.
Response of superficial proximal convoluted tubule to decreased and increased renal perfusion pressure. In vivo microperfusion study in rats.
Circ Res
66:
1184-1189,
1990[Abstract].
15.
Kurihara, N,
Alfie ME,
Sigmon DH,
Rhaleb NE,
Shesely EG,
and
Carretero OA.
Role of nNOS in blood pressure regulation in eNOS null mutant mice.
Hypertension
32:
856-861,
1998
16.
Levine, D,
Iacovitti M,
Burns K,
and
Zhang X.
Real-time profiling of kidney tubular fluid nitric oxide concentrations in vivo.
Am J Physiol Renal Physiol
281:
F189-F194,
2001
17.
Liang, M,
and
Knox F.
Production and functional roles of nitric oxide in the proximal tubule.
Am J Physiol Regul Integr Comp Physiol
278:
R1117-R1124,
2000
18.
Laubach, VE,
Shesely ED,
Smithies O,
and
Sherman PA.
Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death.
Proc Natl Acad Sci USA
92:
10688-10692,
1995[Abstract].
19.
Ling, H,
Gengaro PE,
Edelstein CL,
Martin PY,
Wangsiripaisan A,
Nemenoff R,
and
Schrier RW.
Effect of hypoxia on proximal tubules isolated from nitric oxide synthase knockout mice.
Kidney Int
53:
1642-1646,
1998[ISI][Medline].
20.
Moncada, S,
Palmer RMJ,
and
Higgs EA.
Nitric oxide: physiology, pathophysiology, and pharmacology.
Pharmacol Rev
43:
109-142,
1991[ISI][Medline].
21.
Morrisey, JJ,
McCracken R,
Kaneto H,
Vehaskari M,
Montani D,
and
Klahr S.
Location of an inducible nitric oxide synthase mRNA in the normal kidney.
Kidney Int
45:
998-1005,
1994[ISI][Medline].
22.
Mundel, P,
Bachmann S,
Bader M,
Fischer A,
Kummer W,
Mayer B,
and
Kriz W.
Expression of nitric oxide synthase in kidney macula densa cells.
Kidney Int
42:
1017-1019,
1992[ISI][Medline].
23.
Noris, M,
Morigi M,
Donadelli R,
Aiello S,
Foppolo M,
Todeschini M,
Orisio S,
Remuzzi G,
and
Remuzzi A.
Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions.
Circ Res
76:
536-543,
1995
24.
Preisig, PA.
Luminal flow rate regulates proximal tubule H-HCO
25.
Shesely, EG,
Maeda N,
Kim HS,
Desai KM,
Krege JH,
Laubach VE,
Sherman PA,
Sessa WC,
and
Smithies O.
Elevated blood pressures in mice lacking endothelial nitric oxide synthase.
Proc Natl Acad Sci USA
93:
13176-13181,
1996
26.
Terada, Y,
Tomita K,
Nonoguchi H,
and
Marumo F.
Polymerase chain reaction localization of constitutive nitric oxide synthase and soluble guanylate cyclase messenger RNAs in microdissected rat nephron segments.
J Clin Invest
90:
659-666,
1992[ISI][Medline].
27.
Wang, T.
Nitric oxide regulates HCO
28.
Wang, T.
Role of nitric oxide synthase (nNOS, iNOS and eNOS) in modulating proximal tubule bicarbonate transport and acid-base balance (Abstract).
J Am Soc Nephrol
11:
11A,
2000.
29.
Wang, T,
and
Chan YL.
Mechanism of angiotensin II action on proximal tubular transport.
J Pharmacol Exp Ther
252:
689-695,
1990[Abstract].
30.
Wang, T,
and
Chan YL.
Time and dose dependent effects of protein kinase C on proximal bicarbonate transport.
J Membr Biol
117:
131-139,
1990[ISI][Medline].
31.
Wang, T,
Inglis FM,
and
Kalb RG.
Defective absorption of fluid and bicarbonate in the proximal tubule of mice lacking neuronal nitric oxide synthase (nNOS).
Am J Physiol Renal Physiol
279:
F518-F524,
2000
32.
Wang, T,
Yang C-L,
Schultheis PJ,
Shull GE,
Giebisch G,
and
Aronson PS.
Mechanism of proximal tubule bicarbonate absorption in NHE3 knockout mice.
Am J Physiol Renal Physiol
277:
F298-F302,
1999
33.
Wang, X,
Lu M,
Gao Y,
Papapetropoulos A,
Sessa WC,
and
Wang W.
Neuronal nitric oxide synthase is expressed in principal cell of collecting duct.
Am J Physiol Renal Physiol
275:
F395-F399,
1998
34.
Wilcox, C.
L-Arginine-nitric oxide pathway.
In: The Kidney: Physiology and Pathophysiology (3rd ed.), edited by Seldin D,
and Giebisch G.. Philadelphia, PA: Lippincott Raven, 2000, p. 849-871.