Role of iNOS and eNOS in modulating proximal tubule transport and acid-base balance

Tong Wang

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our laboratory has previously shown that mice lacking neuronal nitric oxide synthase (nNOS) are defective in fluid absorption (Jv) and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (JHCO3) in the proximal tubule and develop metabolic acidosis. The present study examined the transport of fluid and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in the proximal tubule and acid-base status in mice lacking two other isoforms of NOS, inducible NOS (iNOS) and endothelial NOS (eNOS). Proximal tubules were microperfused in situ in wild-type and NOS knockout mice by methods previously described (Wang T, Yang C-L, Abbiati T, Schultheis PJ, Shull GE, Giebisch G, and Aronson PS. Am J Physiol Renal Physiol 277: F298-F302, 1999). [3H]inulin and total CO2 concentrations were measured in the perfusate and collected fluid, and net Jv and JHCO3 were analyzed. These data show that JHCO3 was 35% lower (71.7 ± 6.4 vs. 109.9 ± 7.3 pmol · min-1 · 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<UP><SUB>3</SUB><SUP>−</SUP></UP> transport, whereas eNOS does not directly modulate Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in the kidney proximal tubules.

nitric oxide synthase; inducible nitric oxide synthase; endothelial nitric oxide synthase; neuronal nitric oxide synthase; knockout mice; kidney tubule; transport


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (JHCO3) and Na+ absorption in the proximal tubule are significantly reduced in nNOS knockout mice and that these animals develop a metabolic acidosis (31). These results indicate that the endogenous NO enhances fluid absorption (Jv) and JHCO3 and that nNOS regulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport and acid-base balance. The present study examined Jv and JHCO3 in the proximal tubule and the acid-base status in iNOS and eNOS knockout mice. The effect of the selective iNOS inhibitor L-N6-(1-iminoethyl) lysine (L-NIL) (8) on Jv and JHCO3 in proximal tubules in wild-type and iNOS knockout mice was also examined to evaluate the role of these NOS isoforms in the regulation of renal fluid, Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport, and acid-base balance.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 NaHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 4 KCl, 1 CaCl2, 5 Na-acetate, 5 glucose, 5 L-alanin, 2.5 Na2HPO4, and 0.5 NaH2PO4. The solution was bubbled at room temperature with 5% CO2-95% O2 before use. pH was adjusted to 7.4 with a small amount of NaOH or HCl as required. After the experiment, the perfused tubules were filled with high-viscosity Microfil (Canton Bio-Medical Products, Boulder, CO). The kidney was partially digested in 20% NaOH, silicone rubber casts of the tubule segments were dissected, and the tubular length was measured.

Measurement 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
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES

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 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport, the rate of Jv and JHCO3 in the two isozymes knockout mice was examined. The rates of Jv and JHCO3 in proximal tubules in iNOS and eNOS knockout mice were studied by microperfusion of proximal tubules in vivo. As shown in Table 1 and Fig. 1, JHCO3 and Jv were 35 and 38% lower, respectively, in the iNOS knockout mice. JHCO3 was 71.7 ± 6.4 vs. 109.9 ± 7.3 pmol · min-1 · 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<UP><SUB>3</SUB><SUP>−</SUP></UP> and pH (31). The results suggest that iNOS plays a role in the modulation of JHCO3 in proximal tubules.

                              
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Table 1.   Fluid and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in proximal tubules in wild-type and iNOS and eNOS null mice



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Fig. 1.   Proximal tubule absorption of fluid (Jv) and of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (JHCO3) in wild-type (+/+) and inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) knockout mice (-/-).

In contrast, JHCO3 and Jv did not change significantly in eNOS knockout mice; JHCO3 was 93.3 ± 7.9 vs. 110.6 ± 6.18 pmol · min-1 · mm-1 and Jv was 1.56 ± 0.17 vs. 1.55 ± 0.16 nl · min-1 · mm-1, respectively.

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 · min-1 · 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.


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Fig. 2.   Effects of iNOS inhibitor L-N6-(1-iminoethyl) lysine (L-NIL) on Jv and JHCO3 in wild-type and iNOS knockout mice.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration in wild-type and knockout animals. As shown in Fig. 3, arterial blood HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration and pH did not change significantly in either iNOS or eNOS knockout mice, indicating that the absence of iNOS or eNOS did not affect acid-base balance. Although JHCO3 was 35% lower in iNOS knockout mice than in wild-type control animals, this reduction in proximal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport did not influence blood pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, indicating that the reduction of JHCO3 in the proximal tubule is compensated and balanced in normal acid-base status in iNOS knockout mice.


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Fig. 3.   Comparison of blood pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in iNOS and eNOS knockout mice and control mice. There are no significant (NS) differences in blood pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in either iNOS or eNOS knockout mice.

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.

                              
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Table 2.   Blood pressure and plasma electrolyte in control and NOS knockout mice

Mean arterial blood pressures are also summarized in Table 2. Blood pressure was slightly reduced in iNOS knockout mice, but this reduction did not reach statistical significance. In contrast, blood pressure was significantly higher in eNOS knockout than control mice, similar to previous results (15, 25). Thus iNOS and eNOS differ in the modulation of blood pressure.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption is 70 and 60% lower, respectively, than in wild-type mice (31). nNOS and eNOS have fundamentally opposite effects in modulation of blood pressure (15). eNOS decreases blood pressure by vascular effects, whereas nNOS increases blood pressure by modulation of Na+ transport in the kidney. The present study found that iNOS knockout mice have 38 and 35% lower Jv and JHCO3, respectively, in the proximal tubule and that the iNOS inhibitor decreased 33 and 39% of Jv and JHCO3, respectively, in the wild-type, but not in the knockout, mice. eNOS knockout mice have hypertension without changes in baseline Na+ absorption and JHCO3 in the proximal tubule. These data suggest that iNOS plays a role in upregulation of proximal tubule transport and eNOS does not directly modulate Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations are higher in both iNOS and nNOS knockout mice (28, 31). These results are most likely related to the observation that the rates of JHCO3 in the proximal tubule are lower in both nNOS and iNOS knockout mice. Two cellular mechanisms of JHCO3 modulation in the apical membrane of the proximal tubule, Na+/H+ exchange and/or H-ATPase, may be involved in mediating the effect of NO. Our laboratory previously demonstrated that NO stimulates both Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport in the rat proximal tubule by increasing Na+/H+ exchange (27). The present demonstration that both Jv and JHCO3 are lower in the iNOS knockout mice suggests that Na+/H+-exchange activity is reduced in the absence of iNOS.

Several factors may alter the rate of proximal tubule Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption. These include a change in renal hemodynamics by increasing blood pressure, a change in tubular Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration, or a change in extracellular pH (2, 3, 5-7). Unlike free-flow micropuncture, the in situ microperfusion technique allowed consistent control of the tubular flow rate, Na+, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations, osmolalities, and extracellular pH. Therefore, the volume of Jv and JHCO3 is the result of measured Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport activity in the tubule. Mice with a disruption of the eNOS gene have higher blood pressure, elevated plasma renin concentration, and reduced heart rate despite a modest decrease in kidney renin mRNA (11). Those changes may indirectly change the rate of proximal tubule Na+ absorption and JHCO3. However, under these experimental conditions, Jv and JHCO3 are similar in wild-type and eNOS knockout mice. This result shows that the transport activity of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> does not change significantly in proximal tubules of eNOS null mice, suggesting that eNOS does not directly alter proximal tubule transport of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. However, the changes in renal perfusion pressure by increased blood pressure in eNOS null mice may alter both glomerular filtration rate and capillary pressure and subsequently may alter passive transport by changing tubular permeability or basolateral ion transport activity. It was reported that higher renal perfusion pressure decreased, and lower perfusion pressure increased Jv or JHCO3 (13, 14). Such changes should be possible to detect under these experimental conditions unless both active transport from the apical side and passive transport and/or basolateral transport activity change and mask each other in eNOS null mice. This possibility cannot be excluded in the present study.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> transport: a stimulatory pathway for relatively low concentrations of NO (in the µM range) and an inhibitory pathway for high concentrations (in the mM range) of NO and cGMP (4, 9, 10, 17, 34). Using direct measurement of NO concentrations in kidney tubules in vivo, recent studies demonstrated that the mean NO concentration is 110 nM in both early and late proximal tubules (16). This concentration is similar to that used in our laboratory's previous microperfusion studies in which stimulation of proximal tubule Na+ absorption and JHCO3 was found (27). These observations support the view that NO upregulates proximal tubule transport under physiological conditions. Although it is not clear what fraction of total NO is produced by individual NO isozymes, the acute inhibition of iNOS by a selective inhibitor and knockout of iNOS reduced Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption, indicating that iNOS may contribute to the NO produced in the proximal tubule and regulate Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport activity.


    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.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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