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Am J Physiol Renal Physiol 285: F1160-F1167, 2003. First published September 9, 2003; doi:10.1152/ajprenal.00077.2003
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Nitric oxide inhibits superoxide-stimulated urea permeability in the rat inner medullary collecting duct

Joseph Zimpelmann, Ningjun Li, and Kevin D. Burns

Department of Medicine, Ottawa Hospital, and the Kidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada K1H 7W9

Submitted 24 February 2003 ; accepted in final form 31 July 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The inner medullary collecting duct (IMCD) contains relatively high nitric oxide (NO) synthetic capacity, but the effect of NO on IMCD transport remains unclear. We determined the effect of NO on basal and vasopressin (AVP)-stimulated urea (Purea) and water (Pf) permeabilities in isolated, perfused rat IMCD. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased cGMP production in IMCD, but neither SNAP (10–4 M) nor 8-BrcGMP (10–4 M), the cell-permeable analog of cGMP, affected basal or AVP-stimulated Purea. The free radical superoxide is produced by oxidases in the kidney and can interact with NO. To determine the effect of superoxide generation on transport, IMCDs were incubated with diethyldithiocarbamate (DETC; 10–3 M), the inhibitor of superoxide dismutase (SOD). DETC significantly increased basal and AVP-stimulated Purea (control: 28.7 ± 4.5 vs. DETC: 40.9 ± 6.2 x 10–5 cm/s; P < 0.001; n = 9). Preincubation of IMCD with SNAP or the SOD mimetic tempol completely inhibited DETC-stimulated Purea. DETC caused a significant increase in superoxide generation by IMCD, and this was blocked by SNAP. Incubation of IMCD with the NO synthase (NOS) substrate L-arginine blocked the stimulatory effect of DETC on Purea, and this was reversed by the neuronal NOS inhibitor 7-nitroindazole. In contrast, neither basal nor AVP-stimulated Pf was affected by NO donors or DETC. In summary, exogenous or endogenously produced NO does not affect basal urea transport in the IMCD but inhibits superoxide-stimulated Purea. In the inner medulla, superoxide generation by local oxidases may stimulate urea transport, and the role of endogenous NO may be to dampen this effect by decreasing superoxide levels.

vasopressin; water permeability; nitric oxide synthase; cGMP

ALL THREE ISOFORMS OF THE enzyme nitric oxide synthase (NOS) are expressed in the mammalian kidney, and studies indicate that the gaseous product of NOS, nitric oxide (NO), regulates transport function in several nephron segments (26). In the proximal tubule, both inhibitory and stimulatory effects of NO on apical Na+/H+ exchange have been described (6, 32, 41), and NO appears to inhibit the basolateral Na+-K+-ATPase (16). In thick ascending limb, NO inhibits apical Na+/H+ exchange and Na+-K+-2Cl– cotransport activity (9, 25, 29). Recent studies by Ortiz and Garvin (27, 28) reveal that the bioavailability of NO produced by the thick ascending limb is reduced by endogenous production of superoxide anion (), which itself exerts a stimulatory effect on NaCl transport in this segment. In cortical collecting duct, NO has been shown to inhibit both sodium reabsorption and vasopressin (AVP)-stimulated osmotic water permeability (Pf) (7, 8, 39). Taken together, these studies suggest that NO is an intrarenal natriuretic and diuretic factor.

The inner medullary collecting duct (IMCD) is the final nephron segment and is responsible for fine regulation of net sodium excretion via apical membrane sodium channels, AVP-stimulated water transport via aquaporin water channels, and urea transport via recently described facilitated urea transporters, which contribute to the urine-concentrating mechanism (36). The IMCD contains the highest capacity for NO synthesis of all nephron segments (42) and expresses all three isoforms of NOS (1, 33, 40, 42). However, the function of NO in the IMCD remains unclear. Inhibition of NO production in the inner medulla has been shown to decrease urinary sodium excretion and increase blood pressure in rats (18), suggesting a role for NO in regulating vasa rectae blood flow and/or tubular sodium reabsorption. Zeidel et al. (44) reported that the NO donor sodium nitroprusside (SNP) inhibited ouabain-sensitive oxygen consumption in IMCD, associated with stimulation of cGMP levels, and in suspensions of rabbit IMCD, SNP inhibits sodium uptake (43). Atrial natriuretic peptide (ANP) has also been shown to inhibit both sodium uptake and water transport in IMCD in a cGMP-dependent fashion (17, 24). However, the effects of endogenous or exogenous NO on water or urea transport in the IMCD remain incompletely understood.

The present studies examined the effect of NO on basal and AVP-stimulated urea (Purea) and Pf in the rat IMCD, using the isolated, perfused tubule technique. We uncover a role for an interaction between NO and in the regulation of urea transport in IMCD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of rat IMCDs. Rat terminal IMCD segments were prepared for microperfusion, essentially as described (21). Briefly, male Sprague-Dawley rats (100–150 g), fed regular chow and with unlimited access to water, were killed by decapitation and the kidneys were rapidly removed. Coronal sections were cut and placed in a chilled hypertonic solution containing (in mM) 190 NaCl, 25 NaHCO3, 5 KCl, 10 urea, 5 HEPES, 1.2 MgCl2, 1.5 CaCl2, and 8 glucose, as well as 0.1% albumin (solution A), previously equilibrated with 95% air-5% CO2. IMCD segments were dissected and isolated from the terminal two-thirds of the inner medulla (terminal IMCD). Once dissected, the tubule was transferred to a thermostatically controlled perfusion chamber containing bathing solution, on the stage of an inverted microscope, and mounted on glass pipettes that suspended the tubule in the bath. The temperature of the bath was maintained at 37°C. The inner pipette contained perfusion solution, and perfusion was initiated via a 1-ml syringe connected to PE-10 tubing that was mounted behind the taper at the tip of the perfusion pipette. The perfusate accumulated in the tip of the opposite pipette. Samples of collected perfusate were taken at timed intervals during each experiment. At least 15 min elapsed after each perfusate change before collections were started.

Measurement of Pf. For experiments involving water transport, the bath solution was identical to solution A. The perfusate solution was of identical composition, except that it contained 115 mM NaCl, to ensure existence of a transepithelial osmotic gradient. All solutions were prepared weekly and were equilibrated with 95% air-5% CO2 at 37°C before each experiment, with solution pH between 7.37 and 7.43. For water transport, perfusates contained [methoxy-3H]inulin (100–500 mCi/g, New England Nuclear, Division of PerkinElmer, Boston, MA) that had been dialyzed against distilled water at 4°C for at least 48 h. The bath solution was pumped through the perfusion chamber at 1.0 ml/min. AVP (5 x 10–12 or 10–9 M), NO donors, or other agents were added to the bath as indicated in RESULTS.

Net volume flux (Jv; nl·mm–1·min–1) was calculated for each collection from the equation Jv = (Vo – VL)/L, where Vo is the perfusion rate (nl/min), and L is tubule length (mm). VL was measured directly, and Vo was calculated from Vo = VL(CL/Co), where CL and Co represent [3H]inulin (dpm/nl) of collected fluid and perfusate, respectively. Tubule length was determined by eyepiece micrometer at the end of each experiment. Pf (µm/s) was determined from Pf = RTLP/Vw, where Vw is the partial molar volume of water, and LP is the hydraulic conductivity, determined as described (5): LP = 1/RTSC2B(CB[Vo – VL] + CoVoln{[CB – Co]Vo/[CBVL – CoVo]}). S is the luminal surface area calculated from lumen diameter and assumes that the tubule is a perfect cylinder. Co and CB represent the perfusate and bath osmolalities, respectively, measured with a freezing-point osmometer (Advanced Instruments, VWR, Montreal, Quebec). This calculation of Pf assumes that an effective osmotic gradient exists along the entire length of the perfused tubule. To ensure that this was true for all experimental conditions, tubules were perfused at 17–25 nl/min. In all experiments, three to four collections were made for each experimental condition, and results were averaged to obtain a single value, which was used for statistical analysis.

Measurement of Purea. Purea was measured using a bath-to-lumen urea concentration difference, essentially as described (37, 38). The bath solution contained (in mM) 115 NaCl, 25 NaHCO3, 5 KCl, 5 HEPES, 1.2 MgCl2, 1.5 CaCl2, 8 glucose, and 20 urea. The perfusion solution was identical, except that it contained 20 mM raffinose, but no urea. This established a concentration gradient driving the passive transport of urea from bath to lumen. Urea transport rate was calculated as Jurea = Co·Vo – CL·VL, where Co is the urea concentration in the perfusate, CL is the urea concentration in the collected fluid, Vo is the perfusion rate per unit tubule length, and VL is the collection rate per unit tubule length. Co was zero for all experiments. Purea was then calculated from Jurea, as Purea = Jurea/({pi}·D·{delta}C), where {delta}C is the mean urea concentration difference along the tubule, and D is the tubule inner diameter, measured by eyepiece micrometer. In all experiments involving urea transport, three to four samples were collected for each experimental condition, and results were averaged to obtain a single value that was used for statistical analysis. The tubule characteristics and solution osmolalities for experiments involving urea and water transport are presented in Table 1.


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  		Table 1. Isolated, perfused IMCD characteristics and solution osmolalities

 

The concentrations of urea in the perfusate, bath, and collected fluid were measured by an enzymatic assay, involving spectrophotometry. In this assay, urea is first converted to ammonia in the presence of urease, and then ammonia reacts with alkaline hypochlorite, in the presence of the catalyst phenol nitroprusside to form iodophenol. Iodophenol concentration was measured by absorbance at 570 nm (Spectronic Genesys V, ESBE Scientific, St. Laurent, Quebec) and is proportional to urea concentration. All reagents were purchased as a urea nitrogen assay kit (640A, Sigma, St. Louis, MO). Standard curves were prepared for each experiment, using serial dilutions of the bath solution, which demonstrated linearity.

NO measurements. NO emission from the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and SNP was measured using a carbon fiber NO electrode (ISONOP sensor, 2-mm tip diameter, World Precision Instruments, Sarasota, FL), with calibration performed in the presence of SNAP and copper sulfate, essentially as we have described (14). The incubation solution consisted of an isotonic solution of (in mM) 115 NaCl, 25 NaHCO3, 5 KCl, 5 HEPES, 1.2 MgCl2, 1.5 CaCl2, 8 glucose, and 20 urea.

Measurement of cGMP. Suspensions of rat IMCDs were isolated from renal papillae, after reconstitution in hypotonic solution, exactly as previously described (33). Tubules were then immediately washed three times in Krebs buffer and incubated at 37°C for 30 min in Krebs, supplemented with IBMX (5 x 10–4 M), in the presence or absence of agonists. After addition of ice-cold trichloroacetic acid (final concentration 10% vol/vol), samples were extracted four times with four volumes of water-saturated ether and brought to pH 7.0 with Tris. Aliquots were assayed for cGMP, using a radioligand competitive binding assay kit, containing [3H]cGMP (Amersham, Mississauga, Ontario), as we have performed (32). Results are presented as femtomoles of cGMP per sample, corrected for protein quantity.

Assay of in microdissected IMCD segments. concentrations in microdissected IMCD segments were measured by changes in fluorescence resulting from the oxidation of dihydroethidium (DHE; Sigma) to ethidium, which binds to DNA, producing red fluorescence, as described (15). The assay measures accumulated levels of , rather than instantaneous levels. Briefly, IMCD segments were isolated in solution A and incubated in 95% room air-5% CO2 at 37°C. Thirty minutes after the tubules were loaded with 3 x 10–7 M DHE, tubules were transferred by pipette into 24-well plates (4–5 tubules/well) and incubated with various agonists [diethyldithiocarbamate (DETC) 10–3 M; NADH (10–4 M); or SNAP (10–4 M)]. After a total incubation time of 30 min, the ethidium fluorescence intensity of the tubules in each well was measured using a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Don Mills, Ontario, Canada), with excitation at 490 nm and emission at 610 nm. Images were saved, stored, and quantified, using Northern Eclipse software (Empix Imaging, Mississauga, Ontario). Background fluorescence was subtracted in each experiment, as was the fluorescence at time 0, measured after 30-min incubation with DHE alone.

Statistical analysis. Results are reported as means ± SE. Statistical significance was determined by Student's t-test, in cases of comparison of two groups. For multiple comparisons, one-way ANOVA, following Bonferroni correction, was utilized using SigmaStat software (San Rafael, CA). Significance was assigned at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of NO donors on cGMP in IMCD. NO stimulates guanylate cyclase in many tissues, leading to generation of cGMP. Experiments were first performed to determine whether NO stimulates cGMP production in isolated IMCD segments. As shown in Fig. 1, the NO donor SNAP caused concentration-dependent increases in cGMP in IMCD, with maximal effect at 10–5 M. ANP (10–7 M) also significantly increased production of cGMP. The NO donor SNP also stimulated cGMP production in IMCD segments [control: 11.5 ± 1.0 vs. SNP (10–4 M): 21.1 ± 2.9 fmol/µg; P < 0.04; n = 3]. In separate experiments, NO production was measured in solution, using a carbon fiber NO electrode in the presence of SNAP or SNP. SNAP (10–4 M) was associated with significantly higher NO production in solution, compared with 10–4 M SNP [SNAP (10–4 M): 1.98 ± 0.05 µM NO vs. SNP (10–4 M): 0.23 ± 0.02 µM NO; P < 0.001; n = 3]. Accordingly, further experiments predominantly utilized SNAP (10–4 M) as a NO donor.



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  		Fig. 1. Nitric oxide (NO) stimulates cGMP production in inner medullary collecting duct (IMCD). A: IMCD segments were incubated with S-nitroso-N-acetylpenicillamine (SNAP; 10–4 M) or atrial natriuretic peptide (ANP; 10–7 M) for 30 min before assay of cGMP. *P < 0.01 vs. control (C). **P < 0.001 vs. C (n = 7). B: concentration dependence of stimulation of cGMP by the NO donor SNAP. Values are means ± SE of experiments performed in duplicate. *P < 0.01 vs. C. **P < 0.02 vs. C (n = 3).

 

Effect of NO and 8-BrcGMP on Purea. Single-tubule microperfusion studies were performed to determine the effect of SNAP or the cell-permeable analog of cGMP 8-BrcGMP on urea transport in IMCD. In microdissected IMCD, AVP (5 x 10–12 M) caused a significant increase in Purea (Fig. 2). This concentration of AVP elicited a submaximal response. In separate experiments, AVP (10–9 M) caused a further stimulation of Purea [AVP (5 x 10–12 M): 64.7 ± 1.7 x 10–5 cm/s vs. AVP (10–9 M): 103.2 ± 4.8 x 10–5 cm/s; P < 0.001; n = 3].



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  		Fig. 2. Effect of SNAP and 8-BrcGMP on basal and AVP-stimulated urea permeability (Purea). A: results from 7 tubules in which Purea was determined at baseline, after addition of AVP (5 x 10–12 M) to the bath, and after addition of SNAP (10–4 M) with AVP (5 x 10–12 M) to the bath. Values are means ± SE. *P < 0.001 vs. control (C). B: results from 4 tubules in which Purea was measured at baseline (C), after addition of SNAP (10–4 M), and then after addition of AVP (5 x 10–12 M) and SNAP (10–4 M) to the bath. Values are means ± SE. *P < 0.01 vs. C. C: results from 5 tubules in which Purea was measured at baseline (C), after addition of 8-BrcGMP (10–4 M) to the bath, and then after addition of AVP (5 x 10–12 M) and 8-BrcGMP (10–4 M) to the bath. Values are means ± SE. *P < 0.001 vs. C.

 

The addition of SNAP (10–4 M) to the bath did not affect basal or AVP-stimulated Purea, when added before or after AVP (5 x 10–12 M) (Fig. 2, A and B). The addition of 8-BrcGMP (10–4 M) also had no effect on basal or AVP-stimulated Purea (Fig. 2C).

Effect of generation on Purea in IMCD. Recent studies indicate that stimulates tubular sodium transport, an effect that is diminished in the presence of NO (28, 29). In microdissected IMCD, the inhibitor of superoxide dismutase DETC (10–3 M) (28) caused a significant stimulation of basal and AVP-stimulated Purea (basal Purea: control, 28.7 ± 4.5 x 10–5 cm/s; DETC, 40.9 ± 6.2 x 10–5 cm/s; P < 0.001; n = 9) (Fig. 3).



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  		Fig. 3. Effect of the superoxide dismutase inhibitor diethyldithiocarbamate (DETC) on basal and AVP-stimulated Purea in IMCD. A: results from 9 tubules in which Purea was measured at baseline (C) and then after addition of DETC (10–3 M) to the bath. Values are means ± SE. *P < 0.001 vs. C. B: results from 3 tubules in which Purea was measured at baseline (C), after AVP (5 x 10–12 M) was added to the bath, and after addition of DETC (10–3 M). Values are means ± SE. *P < 0.001 vs. C. **P < 0.005 vs. AVP.

 

Preincubation of tubules with SNAP (10–4 M) completely blocked the stimulatory effect of DETC on Purea, whereas removal of SNAP uncovered the DETC-associated increase in Purea (Fig. 4A). The cell-permeable mimetic of superoxide dismutase 4-hydroxytetramethyl-piperidine-1-oxyl (tempol; 10–4 M) (11, 30) did not affect basal Purea but inhibited the stimulatory effect of DETC on Purea, with a small but significant recovery of Purea when tempol was removed from the bath (Fig. 4B).



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  		Fig. 4. SNAP and 4-hydroxytetramethyl-piperidine-1-oxyl (tempol) inhibit DETC-stimulated Purea in IMCD. A: results from 6 tubules in which Purea was measured at baseline (C), after addition of SNAP (10–4 M) to the bath, and then after addition of SNAP (10–4 M) and DETC (10–3 M) to the bath. In the final part of the experiment, SNAP was removed from the bath perfusate, and DETC alone was present, which caused a significant stimulation of Purea. Values are means ± SE. *P < 0.001 vs. C. B: results from 5 tubules in which Purea was measured at baseline (C), after addition of the permeable superoxide dismutatse mimetic tempol (10–4 M) to the bath, and then after addition of tempol (10–4 M) and DETC (10–3 M) to the bath. In the last part of the experiment, tempol was removed from the bath perfusate, and DETC alone was present. Values are means ± SE. Removal of tempol in the presence of DETC caused a significant stimulation of Purea. *P < 0.005 vs. C.

 

Effect of NO on DETC-induced generation in IMCD. In microdissected IMCD segments, DETC (10–3 M) caused a significant increase in generation of (Fig. 5). The NO donor SNAP (10–4 M) did not affect basal levels but completely inhibited DETC-stimulated production. Incubation of IMCD with cGMP (10–4 M) or 8-BrcGMP (10–4 M) had no effect on generation (control: 15.9 ± 2.1 vs. cGMP: 14.0 ± 1.4, vs. 8-BrcGMP: 17.0 ± 1.1 arbitrary units; P = NS for all comparisons; n = 3–5).



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  		Fig. 5. SNAP inhibits DETC-stimulated superoxide generation in IMCD. Superoxides were measured in isolated IMCD segments by fluorescence emission resulting from the oxidation of dihydroethidium, as described in MATERIALS AND METHODS. IMCD segments were incubated with DETC (10–3 M) and/or SNAP (10–4 M) before measurement of fluorescent units. Incubation of tubules with NADH (10–4 M) was used as a positive control. Values in parentheses above the bars represent nos. of individual tubules. *P < 0.001 vs. C.

 

Effect of endogenous NO production on DETC-stimulated Purea. The IMCD has been shown to have considerable capacity to synthesize NO (42), and the neuronal isoform of nitric oxide synthase (nNOS) is highly expressed in this segment (33). To determine whether endogenous NO production might affect DETC-stimulated urea transport, microdissected IMCD were preincubated with the NOS substrate L-arginine (L-Arg, 0.25 x 10–3 M). As shown in Fig. 6A, L-Arg had no effect on basal Purea, but it blocked DETC-stimulated Purea (control: 21.5 ± 6.2 vs. L-Arg: 22.7 ± 7.7, vs. L-Arg+DETC: 24.9 ± 7.4 x 10–5 cm/s; P = NS for all comparisons; n = 4). Preincubation of IMCD with the inhibitor of nNOS 7-nitroindazole (7-NI; 10–6 M) significantly reversed the inhibitory effect of L-Arg on DETC-stimulated Purea (Fig. 6B).



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  		Fig. 6. Effect of L-arginine (L-Arg) and 7-nitroindazole (7-NI) on DETC-stimulated Purea in IMCD. A: results from 4 tubules in which Purea was measured at baseline (C), after addition of L-Arg (0.25 x 10–3 M) to the bath, and then after further addtion of DETC (10–3 M). Values above the lines represent means ± SE. There was no significant difference among any of the groups. B: results from 3 tubules in which Purea was measured at baseline (C), after addition of 7-NI, the inhibitor of neuronal nitric oxide synthase (10–6 M), after addition of L-Arg (0.25 x 10–3 M), and finally after addition of DETC (10–3 M). Values above the lines represent means ± SE. *P < 0.01 vs. C, P < 0.02 vs. 7-NI, P < 0.01 vs. 7-NI+L-Arg.

 

Effect of NO and DETC on basal and AVP-stimulated Pf. Administration of AVP to the bath caused a concentration-dependent stimulation of Pf, as expected [control: 64.5 ± 19.7 µm/s vs. AVP (5 x 10–12 M): 1,025.4 ± 47.5 µm/s; P < 0.001 vs. control; vs. AVP (10–9 M): 1,349.0 ± 26.9 µm/s; P < 0.005 vs. AVP (5 x 10–12 M); n = 3]. Neither SNAP (10–4 M) nor SNP (10–4 M) affected basal or AVP-stimulated Pf (Fig. 7A). Addition of SNAP (10–4 M) after administration of AVP to the bath also had no significant effect on Pf (Fig. 7B). Similarly, addition of 8-BrcGMP (10–4 M) to the bath had no effect on basal or AVP-stimulated Pf [control: 42.5 ± 13.9 µm/s vs. 8-BrcGMP: 34.2 ± 5.9 µm/s; P = NS vs. control; 8-BrcGMP+AVP (5 x 10–12 M): 1,116.4 ± 17.2 µm/s; P < 0.001 vs. AVP; n = 3]. In contrast, as a positive control, addition of endothelin (10–8 M) to the bath caused a significant inhibition of AVP-stimulated Pf, as previously described [control: 23.8 ± 9.5 µm/s vs. AVP (5 x 10–12 M): 1,211.1 ± 102.8 µm/s; P < 0.001 vs. control; AVP+endothelin (10–8 M): 574.1 ± 51.8 µm/s; P < 0.01 vs. AVP alone; n = 3] (22).



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  		Fig. 7. Effect of NO on basal and AVP-stimulated water permeability (Pf). A: IMCD tubules were incubated with or without SNAP or SNP (10–4 M) in the bath, followed by AVP (5 x 10–12 M) in the bath in the presence or absence of SNAP or SNP. Values in parentheses above the bars indicate nos. of tubules. *P < 0.001 vs. C. B: osmotic Pf was determined in IMCD tubules (n = 4) at baseline (C), after the addition of AVP (5 x 10–12 M), and then after the addition of SNAP (10–4 M) with AVP to the bath. Values are means ± SE at each point. *P < 0.001 vs. C.

 

In contrast to the stimulatory effects of DETC on Purea, DETC had no effect on basal or AVP-stimulated Pf (Fig. 8).



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  		Fig. 8. Effect of DETC on basal and AVP-stimulated osmotic Pf in IMCD. A: results from 4 tubules (results from 2 tubules are superimposed) in which Pf was measured at baseline (C), after addition of DETC (10–3 M) to the bath, and then after addition of AVP (5 x 10–12 M) to the bath. Values are means ± SE. *P < 0.001 vs. C. B: results from 4 tubules in which Pf was measured at baseline (C), after addition of AVP (5 x 10–12 M) to the bath, and and then after addition of DETC (10–3 M) to the bath. Values are means ± SE. *P < 0.001 vs. C.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study examined the effects of NO on urea and water transport in the rat terminal IMCD, using the isolated, perfused tubule technique. Accumulation of urea in the renal medulla is an important determinant of the ability of the kidneys to conserve water. The terminal two-thirds of the IMCD contains only principal cells, which have increased basal permeability to both water and urea, compared with cells of the initial one-third of the IMCD (37). AVP stimulates both water and urea transport in the rat terminal IMCD (38). Facilitated urea transport appears to occur via specific urea transport proteins of the UT-A class, whereas sodium-dependent secondary active urea transport mechanisms have also been described (34, 35). The phosphorylation of UT-A1 in the IMCD is increased acutely by AVP (43), whereas its protein abundance is increased when urine-concentrating ability is reduced (35).

In the present studies, we found no significant effect of endogenous or exogenous NO on basal or AVP-stimulated Purea or Pf in the IMCD. The major positive finding is that NO completely blocked the stimulatory effect of DETC, an inhibitor of superoxide dismutase (SOD), on Purea. This occurred with exogenous NO (SNAP) or with incubation with the NO precursor L-Arg. We utilized SNAP as a NO donor for most experiments, because it was associated with significantly higher NO release than SNP. Although SNAP may release both NO and , incubation of IMCD with SNAP had no effect on accumulated levels of , and indeed, SNAP completely blocked the stimulatory effect of DETC on levels. The data suggest that rapidly activates urea transport in IMCD, and NO may reduce the availability of by combining with to form peroxynitrite. However, the possible independent effects of peroxynitrite on urea transport were not addressed in this study and require further investigation. Our data also confirm that NO stimulates cGMP production in IMCD (13) and that basal Purea in terminal IMCD is relatively high and increased further by AVP (37, 38).

Of all nephron segments, the capacity to generate NO is highest in the IMCD (42), and indeed all three isoforms of NOS are expressed in the IMCD (1, 33, 40, 41). A high-salt diet increases protein expression of nNOS in IMCD (33) and, in cultured mouse IMCD cells, shear stress increases NO production (3). Studies support a role for IMCD-derived NO in modulating sodium transport in IMCD. Zeidel and colleagues (43) showed that SNP inhibited oxygen consumption and sodium uptake in rabbit IMCD cells, an effect mimicked by 8-BrcGMP. Furthermore, cGMP inhibits apical membrane sodium channels in IMCD, an effect that may be involved in mediating the inhibitory effects of ANP on sodium transport in this segment (17).

The present studies focused on effects of NO on urea and water transport, rather than sodium transport. NO had no effect on basal or AVP-stimulated Purea or Pf. Recent studies indicate that tubular segments generate , mainly via the NADH oxidase pathway, with the highest activity in the thick ascending limb of the loop of Henle (15). NAD(P)H oxidase has been localized to various rat nephron segments, including IMCD (4). Reactive oxygen species such as are important regulators of cell signaling and regulate vascular tone, via their interaction with NO. Inactivation of SOD activity with DETC, for example, selectively inhibits NO-induced vasorelaxation in coronary arteries (20). In rats, renal medullary infusion of DETC decreases medullary blood flow and sodium excretion, whereas the SOD mimetic tempol induces the opposite effect (46). In thick ascending limb, endogenous production of is associated with stimulation of Cl– transport and with a decrease in the bioavailability of NO (27, 28). This suggests that regulates nephron transport under physiological conditions.

The present studies demonstrate that DETC significantly increases Purea in IMCD, determined by imposing a bath-to-lumen urea gradient across the tubule, as described elsewhere (37). Exogenous NO blocked this effect, as did incubation of IMCDs with tempol (Fig. 4). The effect of endogenous production of NO in IMCD was also examined. L-Arg had no effect on basal Purea, but it blocked DETC-stimulated Purea (Fig. 6). Furthermore, 7-NI, the inhibitor of nNOS, blocked this inhibitory effect of L-Arg but had no effect on basal Purea. These data suggest that, under basal conditions, levels in IMCD are low and do not affect urea transport. The lack of effect of exogenous NO or the SOD mimetic tempol on basal Purea supports this possibility, because any further decrease in levels induced by these maneuvers would not be expected to affect urea transport. However, stimulation of increases Purea, an effect prevented by NO generation. Accordingly, the data suggest that SOD activity in the inner medulla plays an important role in preventing accumulation of to levels required to stimulate urea transport. In this regard, it is noteworthy that renal medullary SOD activity is markedly reduced in salt-fed Dahl salt-sensitive rats, associated with increased renal oxidative stress (19), and it is conceivable that a reduction in medullary SOD activity in pathophysiological states might induce -mediated stimulation of urea transport. On the other hand, endogenous nNOS activity in IMCD may regulate Purea by decreasing the availability of locally generated . It is also of interest that angiotensin II stimulates Purea in IMCD (12) and increases production by thick limb segments (15). The mechanism whereby stimulates Purea requires further study, including examination of the phosphorylation pattern of UT-A transporters.

ANP stimulates cGMP production in terminal IMCD and inhibits AVP-stimulated osmotic Pf (23, 24). In contrast, Nonoguchi et al. (24) found no effect of ANP on Purea in the rat terminal IMCD (24). In the present studies, we observed no effect of NO on basal or AVP-stimulated Pf (Fig. 7). Experiments utilized submaximal doses of AVP, and, as a positive control, a significant inhibition of AVP-stimulated Pf occurred with endothelin. Our data contrast with reports of inhibitory effects of NO donors on Pf in the rat cortical collecting duct, where activation of guanylate cyclase was proposed to mediate the inhibitory response (7, 8). However, using the excised patch-clamp technique in rat cortical collecting duct, Hirsch et al. (10) found no effect of SNP on cGMP levels or on basal or AVP-stimulated Pf. Furthermore, in our studies addition of 8-BrcGMP, the cell-permeable analog of cGMP, had no effect on Pf. Nonoguchi et al. (23) found that the inhibitory effect of ANP on AVP-stimulated Pf in the terminal IMCD was mimicked by the natural form of cGMP, but not by 8-BrcGMP, each at 10–4 M. They suggested that 8-BrcGMP might induce stimulation of protein kinase A at high concentrations, negating its possible inhibitory effects on Pf (24). Rocha and Kudo (31) found no effect of exogenous cGMP on basal water transport in the rat IMCD, although they observed inhibition of AVP-stimulated Pf. On the other hand, studies in rat kidney slices and renal epithelial cells in culture (LLC-PK1) have shown that exogenous NO stimulates insertion of aquaporin-2 water channels into the apical membrane in a cGMP-dependent fashion, suggesting an increase in Pf (2). It is difficult to reconcile these conflicting data, but our results suggest no net effect of NO on Pf in the rat IMCD. Furthermore, we showed that neither cGMP nor 8-BrcGMP had any effect on production of in IMCD, suggesting that this second messenger pathway is not involved in the inhibitory effect of NO on generation. However, because ANP (10–7 M) caused higher levels of cGMP generation in IMCD compared with SNAP, we cannot rule out the possibility that the lack of effect of SNAP on Pf was due to insufficient stimulation of cGMP.

In summary, enhanced generation of is associated with a stimulation of urea transport but not Pf in the IMCD. While NO does not affect basal Purea or Pf, endogenous or exogenous NO prevents the stimulatory effect of increased levels on Purea. This suggests that nNOS-derived NO in the IMCD may dampen stimulation of urea transport in states of high generation.


   DISCLOSURES
 
This work was supported by a grant (to K. D. Burns) from the Canadian Institutes of Health Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. D. Burns, Div. of Nephrology, The Ottawa Hospital and Univ. of Ottawa, 1967 Riverside Dr., Rm. 535A, Ottawa, Ontario, Canada K1H 7W9 (E-mail: kburns{at}ottawahospital.on.ca).

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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