PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Hypertension. Author manuscript; available in PMC 2010 August 1.
Published in final edited form as:
PMCID: PMC2771701
NIHMSID: NIHMS131370

A NOVEL AMILORIDE SENSITIVE H+ TRANSPORT PATHWAY MEDIATES ENHANCED SUPEROXIDE PRODUCTION IN THICK ASCENDING LIMB OF SALT-SENSITIVE RATS, NOT Na+/H+ EXCHANGE

Abstract

It has previously been reported that H+ efflux via Na+/H+ exchange stimulates NAD(P)H oxidase dependent O2 production in medullary thick ascending limb. We have recently demonstrated that N-methyl-amiloride sensitive O2 production is enhanced in thick ascending limb of salt-sensitive SS rats suggesting that H+ efflux through Na+/H+ exchangers may promote renal oxidative stress and the development of hypertension in these animals. In the current study we demonstrate, using selective and potent inhibitors, that inhibition of Na+/H+ exchange does not mediate the ability of N-methyl-amiloride to inhibit thick ascending limb O2 production. To determine the mechanism of action of N-methyl-amiloride, we examined H+ efflux and O2 production in SS and SS.13BN thick ascending limb of pre-hypertensive, 0.4% NaCl fed rats. Tissue strips containing medullary thick ascending limb were isolated from male SS and salt-resistant consomic SS.13BN rats, loaded with either DHE or BCECF, and imaged in a heated tissue bath. In Na+ replete media, activation of Na+/H+ exchange using an NH4Cl prepulse did not stimulate thick ascending limb O2 production. In Na+ free media containing BaCl2 in which Na+/H+ activity was inhibited, a NH4Cl pre-pulse stimulated mTAL O2 . This response was enhanced in mTAL of SS rats (slope ΔEth/ΔDHE=0.029±0.004) compared to SS.13BN rats (slope=0.010±0.004; p<0.04) and could be inhibited by N-methyl-amiloride (slope=0.005±0.002 & 0.006±0.002 for SS and SS.13BN, respectively). We conclude that only H+ efflux through a specific, as yet unidentified, amiloride-sensitive H+ channel promotes O2 production in medullary thick ascending limb, and that this channel is up-regulated in SS rats.

Keywords: amiloride, blood pressure, free radicals, H+ transport, kidney, NAD(P)H oxidase, pH

Introduction

Superoxide (O2 ) production is enhanced in the outer medulla of Dahl salt-sensitive (SS) rats and has been demonstrated to contribute to the development of hypertension in these animals1, 2. In a recent study we demonstrated that O2 production in response to cellular shrinkage was enhanced in medullary thick ascending limb renal tubular segments (mTAL) of SS rats and that N-methyl-amiloride reduced O2 production in SS mTAL to levels observed in salt-resistant control SS.13BN rats3. These findings were consistent with previous findings indicating that amiloride sensitive O2 production in mTAL could be driven by H+ efflux 4. Together, these data led us to hypothesize that much of the O2 production in mTAL is linked to the activity of Na+/H+ exchange (NHE). To test this hypothesis further, in the current study we utilized unique derivatives of amiloride capable of selective inhibition of NHE-1 and NHE-3, the predominant isoforms of NHE found in mTAL5. Surprisingly, incrementing bath [NaCl] from 154- to 254- to 500mM as we had done in our previous study 3, in the presence of the potent and specific inhibitors of NHE-1 and NHE-3 did not reduce O2 production in mTAL of SS rats to control levels, indicating that the NHE activity was not mediating extracellular NaCl induced O2 production.

Given this data along with the evidence that H+ efflux stimulates mTAL NAD(P)H oxidase4, we hypothesized that N-methyl-amiloride was inhibiting O2 production in SS mTAL by blocking a H+ transport pathway other than Na+/H+ exchange. To test this hypothesis, in the current study we acidified freshly isolated mTAL from SS and SS.13BN rats using the NH4Cl prepulse technique. O2 responses and the rate of pH recovery in mTAL were determined in 1) NaHCO3 free, Na+ replete media in, which the majority of H+ efflux occurs via NHE; 2) Na+ free media in which NHE was inhibited and H+ efflux must occur predominantly through secondary H+ transport pathways other than NHE; and 3) Na+ free media in the presence of Ba2+ in which many of these secondary H+ efflux pathways which are not sensitive to amiloride were inhibited. We hypothesized first that only specific activation of a subgroup of H+ transport pathways other than NHE would result in O2 production in response to H+ efflux in mTAL and that this pathway would be sensitive to inhibition by N-methyl-amiloirde. Second, we hypothesized that this novel pathway of amiloride-sensitive O2 production was linked to the activity of NAD(P)H oxidase and would be enhanced in the mTAL of SS rats compared to salt-resistant SS.13BN rats, thus potentially accounting for the oxidative stress and salt-sensitivity observed in SS rats.

Methods

Experimental animals

Studies used 7–10 week old Male SS and SS.13BN rats (MCW inbred strains 6, 7 weighing 250–350g maintained ad libitium on water and a standard pellet diet containing 0.4% NaCl since weaning in the animal resource center of the Medical College of Wisconsin. All protocols were approved by the Institutional Animal Care Committee.

Solutions

Hanks balanced salt solution (HBSS) was purchased from Invitrogen (Invitrogen, Grand Island, NY). Na+ free solution was prepared by adding Choline-chloride (ChCl 154mM to distilled deionized H20. HEPES (20mM; Sigma Co. St Louis, USA) was added to all solutions and the pH adjusted to 7.40. Apocynin, N-methyl-amiloride, choline chloride, nigericin, KR32568, NH4Cl and BaCl2 were purchased from Sigma Co (Sigma). S3226 and Cariporide were generously provided by Sanofi-Aventis (Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany). Dihydroethedium (DHE) and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF) were purchased from Molecular probes (Molecular Probes, Eugene, OR).

Determination of mTAL superoxide production and pHi

Rats were anesthetized with sodium pentobarbital (60mg/kg/i.p) and isolation of mTAL tissue strips performed as described previously 8 and thin tissue strips containing mTAL placed on a glass coverslip coated with the tissue adhesive Cell-Tak (BD Biosciences, Bedford, MA) for fluorescence imaging. Tissue strips containing mTAL were loaded with either DHE (50mmol/L) or BCECF (6µmol/L) in HBSS for 1 hour at room temperature. Loading buffer was then replaced with HBSS and tissues rested for a further 15 min before being imaged. Coverslips were placed on a heated imaging chamber maintained at 37°C (Warner Instruments, Hamden, CT) that allowed the rapid exchange of superfusion buffer, and mounted on the stage of an inverted microscope.

Fluorescence measurements were made using a Nikon TE2000 inverted microscope with a X60 water immersion (numerical aperture 1.2) objective lens. The signal was detected using a high-resolution digital camera (Photometrics Cascade 512B, Roper Scientific, Tucson, AZ). Excitation was provided by a Sutter DG-4 175W xenon arc lamp (Sutter Instruments, Novato, CA) that allowed high-speed excitation wavelength switching.

Five to ten mTAL epithelial cells were selected within each tissue strip to quantify changes in fluorescent intensity of dyes using Metafluor imaging software (Universal Imaging, Downingtown, PA). BCECF was excited at 440/10 and 490/10. A 510/40-nm band pass emission filter was used to collect BCECF fluorescent signal at 3 second intervals. Intracellular pH ([pHi]) was calibrated in situ at the end of each experiment using a two-point calibration curve by exchanging the bath solution with saline solution containing nigericin (10uM) and KCl (140mM) of known pH9.

Due to the overlap in excitation and emission wavelengths of BCECF and DHE, O2 responses were determined in separate mTAL. A 445/40-nm and a 605/55-nm band pass emission filter were used to collect DHE (380/40X-445/40E) and Eth (480/40X-605/55E) signals. A Lambda-10-3 and rapid filter wheel changer (Sutter Instruments) was used to collect emission signals form DHE and Eth at 3 second intervals. Background Eth and DHE fluorescent signals were subtracted from the average intensity all regions of interest containing mTAL epithelial cells. DHE and Eth signals were then normalized so that the ratio Eth/DHE at time=0 was equal to 1. The change in the ratio of Eth/DHE fluorescent signal across the duration of the experiment was then used as an index of O2 production.

Experimental Protocols

NaCl stimulation of mTAL O2 production

O2 production was stimulated in mTAL of SS and SS.13BN rats by increasing bath NaCl concentration through 154- 254- and 500mM, for 200 seconds at each increment, over a 600 second period, as previously reported 3. O2 responses to incrementing bath NaCl concentration were determined in response to incrementing bath NaCl in the presence of KR32568 ([5-(2-Methyl-5-fluorophenyl)furan-2-ylcarbonyl]guanidine (100µM), a selective inhibitor of NHE-1 (IC50 0.23µM)) 10 and dual inhibition by Cariporide (100µM) and S3226 (100µM), which are selective inhibitors of NHE-1 (IC50 0.033µM)11 and NHE-3 (IC50 0.23µM)12, respectively.

NH4Cl prepulse

To identify the source of amiloride sensitive O2 production in mTAL of SS and SS.13BN rats, we stimulated H+ efflux in mTAL epithelial cells under a number of conditions using the NH4Cl prepulse method. In brief, this method involved adding 20mM of NH4Cl solution to a bath containing tissue strips. As mTAL are highly permeable to NH3 but not NH4 + and this solution contains NH4 + and NH3 in equilibrium, NH3 preferentially enters the mTAL epithelial cells resulting in rapid intra-cellular alkalinization (Figure 2). mTAL were left to bath in this solution for 5–10 minutes in which time pHi returned towards baseline levels. Once pHi reached a stable plateau, the NH4Cl solution was quickly replaced with a vehicle solution which results in rapid acidification of the cell. The rate of recovery of pHi toward baseline levels and the production of O2 following acidification was then recorded.

Figure 2
Representative example of NH4Cl prepulse experiment in mTAL measuring pHi and superoxide production. x-axis, time (seconds); y-axis (left) O2 production assessed as the ratio Eth/DHE fluorescents (arbitary units), (right) intracellular pH (pH ...

Eight protocols were performed in total. Four in which pHi responses to an NH4Cl prepulse were recorded in mTAL loaded with BCECF and four identical protocols in which O2 responses were recorded in mTAL loaded with DHE. Responses in mTAL from SS and SS.13BN rats were compared within each protocol. In one group, an NH4Cl prepulse was performed in bicarbonate free saline (154mM) to stimulate NHE in mTAL. In a second group, an NH4Cl prepulse was performed in the same media with the exception that NaCl was replaced with ChCl to produce Na+ free media to inhibit NHE activity. In a third group, an NH4Cl prepulse was performed in Na+ free media in the presence of BaCl2 (10mM). BaCl2 was added as this ion has been previously demonstrated to inhibit amiloride-insensitive H+ flux in mTAL13. The final group contained the same media as the third group with the addition of 100µM N-methly-amiloride. In some mTAL apocynin (100µM) was added to the bath 30 min prior to stimulation by NH4Cl prepulse in Na+ free, BaCl2 (10mM) media to determine the contribution of NAD(P)H oxidase.

Angiotensin II stimulation of O2 production

O2 production was stimulated in mTAL of SS and SS.13BN rats by the addition of angiotensin II (1µM) to the bath as previously reported14. O2 responses were determined as the change in ratio of ΔEth/ΔDHE 200 seconds after administration of angiotensin II. To determine whether O2 responses to angiotensin II may be mediated by amiloride-sensitive pathways, O2 responses to angiotensin II were also determined in both SS and SS.13BN rats in the presence of N-methyl-amiloride (100µM).

Data and statistical analysis

Data are expressed as means ± standard error. Responses in mTAL of SS and SS.13BN rats were compared with a 2-way ANOVA using a Bonferroni post-hoc test. For all other data, significance was evaluated using an unpaired t-test. The level required to reach significance was p<0.05.

For addition methods please see http://hyper.ahajournals.org.

Results

NHE inhibitors on O2 response to incrementing bath NaCl

Incrementing bath NaCl concentration through 154- 254- and 500mM increased O2 production in mTAL of both SS and SS.13BN rats. In the presence of KR32568, a selective and potent inhibitor of NHE-110, total O2 production over the 600 second protocol was approximately 40% greater in mTAL of SS rats compared to the responses observed in mTAL of SS.13BN rats (Figure 1; P<0.005). Unlike N-methyl-amiloride administration which we have previously reported to inhibit O2 production and abolished strain differences between mTAL of SS and SS.13BN rats3, our current data indicate that KR32568 did not reduce O2 responses in SS rats to the levels observed in mTAL of SS.13BN animals. A similar response was observed in response to incrementing bath NaCl in the presence of Cariporide and S3226 which are potent inhibitors of NHE-1 (IC50 0.033µM)11 and NHE-312, respectively. In the presence of both Cariporide and S3226 O2 responses to incrementing bath NaCl remained elevated in mTAL of SS rats compared to mTAL of SS.13BN rats (Figure 1; P<0.005). Neither outer medullary protein expression of NHE-1 nor NHE-3 were different between SS and SS.13BN rats (please see http://hyper.ahajournals.org.)

Figure 1
Effect of specific inhibition of NHE isoforms on NaCl induced superoxide production in mTAL of SS and SS.13BN rats in response to incrementing extracellular NaCl concentration. x-axis, rat strain; y-axis; final ratio of ΔEth/ΔDHE (arbitary ...

NH4Cl prepulse

Baseline pHi levels in mTAL of SS and SS.13BN in each bath solution as well as the rates of pHi recovery over the first 20 seconds following cellular acidification by NH4Cl prepulse are given in Table 1. Figure 2 demonstrates pHi and O2 responses during NH4Cl prepulse in Na+ free media with BaCl2. Note that O2 production was only stimulated during H+ efflux following removal of NH4Cl, not during H+ influx.

Table 1
Intracellular pH in SS and SS.13BN mTAL during NH4Cl prepulse

As observed in Figure 3, in Na+ replete media (154mM NaCl, pH 7.40) pHi recovered rapidly in both mTAL from SS and SS.13BN rats following removal of NH4Cl from the bath and cellular acidification. In the absence of bicarbonate, which inhibits Na+/HCO3 exchange, the initial rate of pHi recovery in Na+ replete media can be used as an index of NHE activity9. The initial rate of recovery of pHi in Na+ replete media was not different between mTAL of SS and SS.13BN rats (Figure 3.b). Despite activation of NHE and rapid recovery of pHi, no significant O2 production was observed in response to an NH4Cl prepulse in Na+ replete media in mTAL from either SS or SS.13BN rats (Figure 3.a).

Figure 3
Intracellular pH recovery and superoxide production in mTAL of SS and SS.13BN rats following acidification by NH4Cl prepulse. (a & e) media containing 154mM Na+, (b & f) 0 Na+ (c & g) 0 Na+ + BaCl2 (100mM), (d & h) 0 Na ...

In Na+ free media in which NaCl was replaced with ChCl to inhibit NHE, pHi recovery from an NH4Cl prepulse was less than that observed in Na+ replete media (Table 1). The rate of pHi recovery, however, did not differ between mTAL from SS and SS.13BN rats (Figure 3.d). In Na+ free media, O2 responses were observed in SS and SS.13BN rats corresponding to the time in which H+ efflux was occurring. These O2 responses did not differ however between mTAL from SS and SS.13BN rats (Figure 3.c).

In Na+ free media in which BaCl2 had been added as a non-specific inhibitor of ion transporters15, pHi recovery following cellular acidification was reduced compared to Na+ free media alone (Table 1; P<0.05). Importantly, the rate of recovery of pHi in mTAL of SS rats was greater than that observed in mTAL of SS.13BN rats under these conditions (Figure 3.f; P<0.05). Further, O2 production associated with H+ efflux was significantly greater in mTAL of SS rats compared to mTAL of SS.13BN rats during this period (Figure 3.e; P<0.05). Addition of N-methyl-amiloride reduced the rate of pHi recovery in mTAL of SS rats to similar levels to those observed in mTAL of SS.13BN rats (Figure 3.h) and completely abolished O2 production in response to recovery from cellular acidification (Figure 3.g).

The role of NAD(P)H oxidase

The NAD(P)H oxidase inhibitor apocynin completely abolished O2 production in SS mTAL in response to an NH4Cl prepulse in Na+ free media containing BaCl2 (Figure 4). Addition of angiotensin II (1µM), a well known stimulator of NAD(P)H oxidase, stimulated the production of O2 in mTAL from both SS and SS.13BN rats. Importantly, in response to angiotensin II, O2 production was greater in mTAL of SS rats and O2 production in both SS and SS.13BN rats could be abolished by prior administration of N-methyl-amiloride (100µM) (Figure 5).

Figure 4
Effect of apocynin on superoxide production in mTAL of SS and SS.13BN rats following acidification by NH4Cl prepulse. O2 production during recovery from an NH4Cl prepulse in media containing 0 Na+ + BaCl2 (100mM) incubated with the NAD(P)H oxidase ...
Figure 5
Effect of N-methyl-amiloride on angiotensin II induced superoxide production in mTAL of SS and SS.13BN rats. y-axis, O2 production assessed as the ratio Eth/DHE fluorescents in response to addition of 1µM angiotensin II to the bath perfusate ...

Discussion

The major findings of this study are 1) that N-methyl-amiloride inhibits O2 production in mTAL by inhibiting H+ efflux specifically through a novel, Na+ insensitive H+ transport pathway, not its classical target NHE; 2) that this amiloride-sensitive H+ transport pathway is enhanced in SS rats. Our finding that a novel H+ transport pathway mediates O2 production in renal tubular cells is of particular relevance given the importance of O2 and oxidant stress in cardiovascular and renal disease and represents a significant step forward in our understanding free radical biology in the kidney. The observed up-regulation of this oxidant producing pathway in one of the most commonly used models of hypertension, the Dahl SS rats, indicates that this pathway may contribute to the increased outer medullary oxidative stress and hypertension found in SS animals. Further, our data suggest that amiloride analogues such as N-methly-amiloride may be useful inhibitors of this pathway and therefore of clinical relevance in diseases where oxidant stress is implicated, such as salt-sensitive hypertension.

Sodium stimulation of O2 in the presence of specific NHE inhibitors

In a previous study we demonstrated that O2 production in mTAL in response to cell shrinkage following incrementing bath NaCl was greater in mTAL of SS rats than that in mTAL of SS.13BN rats3. This finding was important as we have previously demonstrated that enhanced outer medullary oxidative stress contributes to the development of salt-sensitive hypertension in SS rats2. Importantly, in our previous study N-methyl-amiloride reduced O2 production in SS rats whereby O2 production became equal in SS and salt-resistant control SS.13BN rats in response to cell shrinkage3. We were therefore surprised in the current study when we found that in the presence of the potent NHE-1 inhibitor KR32568, the rate of O2 production remained elevated in SS rats compared to SS.13BN rats in response to incrementing bath [NaCl] from 154- to 254- to 500mM as we had done in our previous study3, Even the simultaneous administration of both Cariporide and S3226 to inhibit both NHE-1 and NHE-3 activity failed to reduce O2 production in mTAL of SS rats which remained elevated above that in SS.13BN rats. Cariporide can inhibit both NHE-2 (IC50 1.6µM) and NHE-111, so dual pharmacological inhibition using Cariporide and S3226 would have inhibited NHE-1, NHE-2 and NHE-3. Since these are the primary isoforms of NHE identified in mTAL5, our data strongly indicated that NHE was not involved in the O2 responses we observed. In light of these data as well as evidence that H+ efflux stimulates mTAL NAD(P)H oxidase, we hypothesized that N-methyl-amiloride was inhibiting O2 production in SS mTAL by blocking a H+ transport pathway other than Na+/H+ exchange.

NH4Cl prepulse studies

To determine whether N-methyl-amiloride may be inhibiting O2 production in SS mTAL by inactivating transport pathways other than NHE, we utilized the NH4Cl prepulse method to activate H+ efflux in SS and SS.13BN rats under a variety of bath conditions. While there are numerous transporters present in mTAL capable of extruding H+, NHE is the most effective at rapidly removing H+ and dominates the pH recovery response following acidification. The rational for inhibiting NHE in the current study was based on the idea that, because NHE dominates H+ extrusion, activation of less sensitive H+ transporters in response to an acid load would be limited and obscured by the ability of NHE to rapidly remove the stimuli. By inhibiting NHE using Na+ free media, the task of removing the H+ from the cell is then left to secondary, Na+ independent transport pathways. In this case, even though the rate of overall H+ efflux would be slowed, more H+ would have to be extruded through these pathways as NHE is inactive. Given that O2 production in response to an NH4Cl prepulse was enhanced by inhibition of NHE, our data demonstrate that H+ efflux through a secondary, Na+ independent H+ pathway was responsible for H+ efflux induced production of O2 .

Only when NHE activity was blocked by reducing media [Na+] to zero, did cellular acidification stimulate significant O2 production. Neither the rate of O2 production nor the rate of recovery of pHi however, were different between mTAL of SS or SS.13BN rats in Na+ free media unless BaCl2 (10mM) was added to the bath. BaCl2 was used in this study to further inhibit H+ transport not associated with amiloride-sensitive O2 production thereby isolating and stimulating O2 producing H+ currents further. We speculated that Ba2+ would inhibit unrelated, but not amiloride sensitive pathways of H+ efflux, since amiloride has been demonstrated previously to inhibit amiloride-insensitive H+ influx in mTAL but not amiloride sensitive H+ influx in Na+ free media13.

In Na+ free media in the presence of BaCl2, pHi recovery was significantly greater in mTAL of SS rats compared to SS.13BN rats. Under these conditions, in response to cellular acidification using an NH4Cl prepulse, O2 production was also significantly greater in mTAL of SS rats compared to mTAL of SS.13BN rats. Importantly, addition of N-methyl-amiloride significantly reduced the rate of pHi recovery in mTAL of SS rats to levels observed in SS.13BN rats. Further, N-methyl-amiloride completely abolished O2 production in Na+ free BaCl2 media response to an NH4Cl prepulse. These data indicate that an amiloride sensitive H+ transport pathway, other than NHE, is present in mTAL and that when activated, this transport pathway mediates the production of O2 . Further, our data indicate that this pathway is enhanced in mTAL of SS rats compared to mTAL of salt-resistant SS.13BN rats.

We were unable to detect N-methyl-amiloride sensitive H+ transport in SS.13BN rats in the current study, suggesting that the pathway(s) identified in SS mTAL may not be active in SS.13BN mTAL. Opposing this conclusion, in Na+ free media containing Ba2+, an NH4Cl prepulse stimulated significant O2 production in mTAL of SS.13BN rats and this could be abolished by addition of N-methyl-amiloride (Figure 3.e & 3.g.). Given these data, we conclude that it is likely that the pathway(s) detected in SS mTAL are also present in mTAL of SS.13BN rats. It would appear however, that these pathways are less active in mTAL of SS.13BN rats and that the level of Na+ independent amiloride-senstive H+ transport in this strain is below detectable limits using the BCECF dye method.

As Ba2+ is thought to inhibit both ROMK and Na+K+ATPase mediated transport15, 16, addition of Ba2+ would likely have dissipated mTAL membrane potential. Importantly, Liu et al have demonstrated that depolarization of macula densa stimulates O2 production, raising the possibility that Ba2+ may have stimulated greater O2 production in our study by dissipating membrane potential17. However, it should be noted that the O2 responses observed in our study occurred only during H+ efflux and were inhibitable by amiloride suggesting H+ efflux through amiloride sensitive channels rather than depolarization mediated the response.

While O2 production was observed in Na+ free media in the absence of BaCl2 in response to cellular acidification, it remains unclear if was due to activation of Ba2+ insensitive transporters at a reduced rate, or a distinct pathway of H+ transport also capable of stimulating O2 . Importantly, only when Ba2+ was present were we able to identify differential pHi and O2 between SS and SS.13BN mTAL indicating a select BaCl2 insensitive subgroup of H+ transporters is likely be responsible for differences in amiloride sensitive O2 production in mTAL of SS and SS.13BN rats.

Physiological relevance of amiloride-sensitive O2 production in mTAL and its relation to salt-sensitive hypertension

Like NHE-1 which is known to be stimulated by both intracellular acidification and cell shrinkage (independent of pHi)5, 18, it appears that the amiloride-sensitive H+ transport pathway that we have begun to characterize in this study can be activated by multiple stimuli. We have demonstrated that cellular shrinkage following increased extracelluar NaCl concentration, angiotensin II and cellular acidification in Na+ free media can all stimulate N-methyl-amiloride sensitive O2 production in mTAL. It is unlikely that cellular acidification would normally act as a primary stimuli to activate mTAL O2 production in vivo given Na+ is present and NHE is active. However, other stimuli such as cell shrinkage or angiotensin II would be expected to stimulate amiloride-sensitive O2 production in vivo in a variety of physiological and pathophysiological condiditions.

While the factors that activate this pathway in vivo remain unclear, importantly, Li et al have demonstrated in the in vivo kidney that greater than 50% of outer-medullary oxidative stress is amiloride-sensitive suggesting that pathways such as that identified in the current study are active. Given these data we speculate that differences in amiloride-senstive H+ transport and O2 production observed between mTAL of SS and SS.13BN rats in the current study may account for differences in outer medullary O2 levels observed between these rat strains in vivo 2, 19.

Our data indicating that in the presence of Na+, cellular acidification did not stimulate mTAL O2 production significantly is in contrast to the results of Li et al who reported that Na+ was required for O2 production following an NH4Cl prepulse 4. The major difference between the present study and that of Li et al is that we performed fluorescent measurements of O2 (DHE) and pHi (BCECF) separately in different tissue strips to avoid the possibility of overlapping fluorescent signals confounding our results. Li et al report that they measured pHi and O2 production in mTAL simultaneously by dual loading mTAL with BCECF and DHE4.

What is the amiloride-sensitive H+ transporter that mediates superoxide production in mTAL?

Numerous transporters are capable of extruding H+ from mTAL epithelial cells. While in the current study we have ruled out a role of NHE in mediating mTAL O2 production, the identity of the amiloride-sensitive H+ transporter mediating enhanced O2 production in SS mTAL remains undetermined. Our data do, however, indicate that the amiloride sensitive H+ current is associated with NAD(P)H oxidase. Many of the subunits of NAD(P)H oxidase are up-regulated in the renal medulla of pre-hypertensive SS rats 2. In the current study, we demonstrate that apocynin, an inhibitor of NAD(P)H oxidase, abolished O2 responses in response to activation of this amiloride-sensitve H+ current by NH4Cl. Furthermore, angiotensin II, which is a well known activator of NAD(P)H oxidase stimulated O2 production in SS mTAL and this could be inhibited by amiloride.

The membrane bound subunit of NAD(P)H oxidase NOX2 has been shown to be associated with voltage-gated H+ channels in immune cells, the role of which appears to be to extrude H+ and act as a charge compensator for the electrogenic generation of superoxide by the oxidase20. Interestingly, these voltage gated H+ channels share a number of traits with that of the amiloride-sensitive H+ transport pathway identified in mTAL in the current study in mTAL. Both can be activated by intracellular acidification, both are uni-directional (only extruding H+), and both are relatively insensitive to BaCl2 21. Further studies will be required to determine the specific molecular target of amiloride in mTAL that mediates inhibition of O2 production.

Perspectives

Many of the traits of salt-sensitive hypertension such as early-stage renal failure which are so pervasive in African Americans are recapitulated in the Dahl salt-sensitive rat 6, 22, 23. O2 has been demonstrated to enhance Na+ reabsorption by NKCC and Na+/H+ exchangers in mTAL 24, 25 and excess Na+ reabsorption by the mTAL has been implicated in the development of salt-sensitive hypertension in a number of human populations, including African Americans 26. In the current study we characterize H+ transport in the mTAL of salt-sensitive and salt-resistant rat strains and identify a novel target for amiloride that, when activated, stimulates the excess production of O2 . We conclude that H+ efflux from mTAL cells that results in O2 production occurs only through a specific, N-methyl-amiloride sensitive transport pathway, not H+ efflux via NHE, and that this novel H+ efflux pathway is enhanced in mTAL of salt-sensitive rats. The potential inhibition of this oxidant producing H+ transport pathway by amiloride analogues merits further research as a potential treatment for renal oxidative stress and salt-sensitive hypertension.

Acknowledgments

Sources of funding

This work was funded by NHBLI grants HL-29587 and HL-82798 and American Heart Association fellowship 0625793Z.

Footnotes

Conflict(s) of Interest/Disclosures

none

References

1. Cowley AW., Jr. Renal medullary oxidative stress, pressure-natriuresis, and hypertension. Hypertension. 2008;52:777–786. [PMC free article] [PubMed]
2. Taylor NE, Glocka P, Liang M, Cowley AW., Jr. NADPH oxidase in the renal medulla causes oxidative stress and contributes to salt-sensitive hypertension in Dahl S rats. Hypertension. 2006;47:692–698. [PubMed]
3. O'Connor PM, Lu L, Schreck C, Cowley AW., Jr. Enhanced amiloride-sensitive superoxide production in renal medullary thick ascending limb of Dahl salt-sensitive rats. Am J Physiol Renal Physiol. 2008;295:F726–F733. [PubMed]
4. Li N, Zhang G, Yi FX, Zou AP, Li PL. Activation of NAD(P)H oxidase by outward movements of H+ ions in renal medullary thick ascending limb of Henle. Am J Physiol Renal Physiol. 2005;289:F1048–F1056. [PubMed]
5. Burckhardt G, Di Sole F, Helmle-Kolb C. The Na+/H+ exchanger gene family. J Nephrol. 2002;15 Suppl 5:S3–S21. [PubMed]
6. PhysGen. PGA. The Medical College of Wisconsin. 2008. Available at: http://pga.mcw.edu.
7. Cowley AW, Jr., Roman RJ, Kaldunski ML, Dumas P, Dickhout JG, Greene AS, Jacob HJ. Brown Norway chromosome 13 confers protection from high salt to consomic Dahl S rat. Hypertension. 2001;37:456–461. [PubMed]
8. Dickhout JG, Mori T, Cowley AW., Jr. Tubulovascular nitric oxide crosstalk: buffering of angiotensin II-induced medullary vasoconstriction. Circ Res. 2002;91:487–493. [PubMed]
9. Kikeri D, Azar S, Sun A, Zeidel ML, Hebert SC. Na(+)-H+ antiporter and Na(+)-(HCO3-)n symporter regulate intracellular pH in mouse medullary thick limbs of Henle. Am J Physiol. 1990;258:F445–F456. [PubMed]
10. Roh HY, Jung IS, Park JW, Yun YP, Yi KY, Yoo SE, Kwon SH, Chung HJ, Shin HS. Cardioprotective effects of [5-(2-methyl-5-fluorophenyl)furan-2-ylcarbonyl]guanidine (KR-32568) in an anesthetized rat model of ischemia and reperfusion heart injury. Pharmacology. 2005;75:37–44. [PubMed]
11. Kleemann HW, Weichert AG. Recent developments in the field of inhibitors of the Na+/H+ exchanger. Idrugs. 1999;2:1009–1025. [PubMed]
12. Schwark JR, Jansen HW, Lang HJ, Krick W, Burckhardt G, Hropot M. S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types. Pflugers Arch. 1998;436:797–800. [PubMed]
13. Amlal H, Paillard M, Bichara M. NH4+ transport pathways in cells of medullary thick ascending limb of rat kidney. NH4+ conductance and K+/NH4+(H+) antiport. J Biol Chem. 1994;269:21962–21971. [PubMed]
14. Mori T, O'Connor PM, Abe M, Cowley AW., Jr. Enhanced superoxide production in renal outer medulla of Dahl salt-sensitive rats reduces nitric oxide tubular-vascular cross-talk. Hypertension. 2007;49:1336–1341. [PubMed]
15. Karim Z, Szutkowska M, Vernimmen C, Bichara M. Recent concepts concerning the renal handling of NH3/NH4+ J Nephrol. 2006;19 Suppl 9:S27–S32. [PubMed]
16. Jans F, Balut C, Ameloot M, Wouters P, Steels P. Investigation of the Ba2+-sensitive NH4+ transport pathways in the apical cell membrane of primary cultured rabbit MTAL cells. Nephron Physiol. 2007;106:p45–p53. [PubMed]
17. Liu R, Garvin JL, Ren Y, Pagano PJ, Carretero OA. Depolarization of the macula densa induces superoxide production via NAD(P)H oxidase. Am J Physiol Renal Physiol. 2007;292:F1867–F1872. [PubMed]
18. Demaurex N, Grinstein S. Na+/H+ antiport: modulation by ATP and role in cell volume regulation. J Exp Biol. 1994;196:389–404. [PubMed]
19. Taylor NE, Maier KG, Roman RJ, Cowley AW., Jr. NO synthase uncoupling in the kidney of Dahl S rats: role of dihydrobiopterin. Hypertension. 2006;48:1066–1071. [PubMed]
20. DeCoursey TE. Voltage-gated proton channels. Cell Mol Life Sci. 2008;65:2554–2573. [PubMed]
21. Byerly L, Suen Y. Characterization of proton currents in neurones of the snail, Lymnaea stagnalis. J Physiol. 1989;413:75–89. [PubMed]
22. Campese VM. Salt sensitivity in hypertension. Renal and cardiovascular implications. Hypertension. 1994;23:531–550. [PubMed]
23. Grim CE, Robinson M. Salt, slavery and survival- hypertension in the African diaspora. Epidemiology. 2003;14:120–122. discussion 124–126. [PubMed]
24. Juncos R, Garvin JL. Superoxide enhances Na-K-2Cl cotransporter activity in the thick ascending limb. Am J Physiol Renal Physiol. 2005;288:F982–F987. [PubMed]
25. Silva GB, Ortiz PA, Hong NJ, Garvin JL. Superoxide stimulates NaCl absorption in the thick ascending limb via activation of protein kinase C. Hypertension. 2006;48:467–472. [PubMed]
26. Burnier M. Ethnic differences in renal handling of water and solutes in hypertension. Hypertension. 2008;52:203–204. [PubMed]