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Hemodynamics and Vascular Regulation
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Ouabain Inhibits Tubuloglomerular Feedback in Mutant Mice with Ouabain-Sensitive α1 Na,K-ATPase

John N. Lorenz, Iva Dostanic-Larson, Gary E. Shull and Jerry B. Lingrel
JASN September 2006, 17 (9) 2457-2463; DOI: https://doi.org/10.1681/ASN.2006040379
John N. Lorenz
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Iva Dostanic-Larson
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Gary E. Shull
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Jerry B. Lingrel
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Abstract

Initiation of tubuloglomerular feedback (TGF) depends on Na-K-2Cl co-transport in the macula densa (MD), but it is less clear whether Na,K-ATPase is responsible for establishing the inward Na+ gradient. It has been proposed that apical colonic H,K-ATPase, perhaps in concert with the Na/H exchanger (NHE2), may account for MD Na+ exit in these cells. This study evaluated TGF responses by micropuncture in mutant mice with altered ouabain sensitivity of the α1 and α2 Na,K-ATPase isoforms. TGF responses in α1-sensitive/α2-resistant mice were inhibited by intravenous ouabain (control stop-flow pressure = 9.7 ± 0.9 versus 1.6 ± 0.5 mmHg with intravenous ouabain). Subsequent inclusion of cyclohexyladenosine (10 μM) in the tubule perfusate confirmed the ability of the afferent arteriole to contract in the presence of ouabain. In α1-resistant/α2-resistant mice, ouabain infusion had no effect on TGF responses. In separate experiments, loop of Henle perfusion with 50 μM ouabain decreased TGF responses (control stop-flow pressure) from 10.5 ± 1.1 to 3.9 ± 1.0 mmHg in α1-sensitive/α2-resistant mice but had no effect in α1-resistant/α2-resistant mice, and afferent arteriole responsiveness again was confirmed by cyclohexyladenosine. TGF responses in NHE2 and colonic H,K-ATPase knockout mice were not different from those of wild-type mice. These data indicate that TGF requires activity of the α1 Na,K-ATPase, presumably in the MD. Furthermore, the data show that neither NHE2 nor colonic H,K-ATPase is essential for initiation of TGF responses.

Tubuloglomerular feedback (TGF) is a mechanism whereby the composition of the tubular fluid in the distal nephron is sensed by the macula densa (MD), which generates a signal-regulating afferent arteriolar tone and GFR. Although it is well established that initiation of TGF depends on activity of the apical Na,K-2Cl co-transporter in the MD, the involvement of other ion transporters in the signaling process remains a matter of investigation (1,2). In this regard, there is debate about whether the MD expresses Na,K-ATPase in sufficient quantities to sustain the necessary electrochemical gradient for other ion transport processes. Studies of ATPase activity using electron probe microanalysis concluded that the MD in dogs, rats, or rabbits does not have ATPase activity (3), and immunolocalization studies revealed only very sparse labeling for Na,K-ATPase in MD cells (4). Using a highly sensitive microenzymatic analysis of ATPase activity in MD that were dissected from rabbit, Schnermann and Marver (5) reported that these cells contain Na,K-ATPase but at very low levels compared with surrounding cells. Therefore, it has been reasoned that the capacity for transcellular solute transport by the MD is low and may reflect its role as a sensing epithelium.

Largely through the work of Bell and Peti-Peterdi and colleagues, the existence of several alternative transport pathways in MD cells has been elucidated. They proposed that MD cells express the colonic H,K-ATPase (cHKA) on the apical membrane that may be the primary efflux pathway for Na+ from these cells. Functioning as a Na(H),K-ATPase and/or in concert with apical Na/H exchange (by NHE2), it was suggested that this pathway is centrally involved in regulating MD [Na+]i and TGF signaling (6). Whereas other studies have confirmed reasonably high expression of cHKA in MD cells (7), the relative expression with respect to the Na,K-ATPase is not yet clear. In contrast to the lack of histochemical evidence for basolateral N,K-ATPase in rabbit (6), these investigators (8) and others (9) have shown demonstrable levels of α1 Na,K-ATPase in rat MD, suggesting the possibility of species heterogeneity.

Further complicating the interpretation of these functional data is our current understanding that the α1 subunit of the Na,K-ATPase in rodents (rats and mice) largely is resistant to ouabain (10). This ouabain-resistance of this predominant renal epithelial isoform has hampered attempts to clarify the role of Na,K-ATPase activity in regulating renal ion transport. Therefore, a central question remains as to whether α1 Na,K-ATPase is expressed in MD cells at sufficient levels to influence TGF signaling. In this regard, gene-targeted mice in which the α1 and α2 NKA isoforms were mutated to alter their ouabain sensitivity were recently generated. In this study, we explored the effects of ouabain on TGF signaling in mice that express either the ouabain-sensitive or the ouabain-resistant α1 Na,K-ATPase isoform. To remove the possibility of confounding influences from the α2 Na,K-ATPase isoform (which is expressed in the renal vasculature), all mice that were used in these experiments expressed a ouabain-resistant α2 Na,K-ATPase isoform. Finally, to explore the possible involvement of other MD transport pathways that are implicated in TGF signaling, we report TGF responses in NHE2 knockout and cHKA knockout mice.

Materials and Methods

Animals

Animals were obtained from established colonies at the University of Cincinnati. Mice that were used for analysis of the role of Na,K-ATPase in TGF signaling were on a mixed 129SvJ and Black Swiss background and included double-mutant mice that express the ouabain-sensitive α1 Na,K-ATPase isoform and the ouabain-resistant α2 Na,K-ATPase isoform (α1S/S/α2R/R) (11); as a control, mice that express the ouabain-resistant α1 Na,K-ATPase isoform and the ouabain-resistant α2 Na,K-ATPase isoform (α1R/R/α2R/R) were used (12). NHE2-deficient mice (NHE2−/−) and littermate controls (NHE2+/+) were derived from heterozygous crosses on a Non-Swiss Albino background (13). Colonic H,K-ATPase knockout (cHKA−/−) and wild-type control (cHKA+/+) mice were on a C57BL/6 background (14). Genotypes were determined by PCR analysis of DNA from tail biopsies, as described (11–14). Experiments were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee at the University of Cincinnati.

General Micropuncture Methods

Na,K-ATPase mutant mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (70 μg/g body wt) and supplemented as needed via an intraperitoneal catheter. In experiments involving NHE2 knockout and cHKA knockouts, mice were anesthetized with intraperitoneal injections of ketamine (50 μg/g body wt) and thiobutabarbital (Inactin; RBI, Natick, MA; 100 μg/g body wt). Body temperature was maintained at 37.5°C using a feedback-controlled, heated surgical table, and animals were provided with 100% O2 to breathe. After tracheostomy, the right femoral artery and vein were cannulated for measurement of BP and infusion of maintenance fluids and drugs. The bladder was cannulated for urine collection. BP, heart rate, and proximal stop-flow pressure (PSF; see below) were monitored using a PowerLab system (ADInstruments, Colorado Springs, CO). The left kidney was exposed via a flank incision, dissected free, placed in a Lucite cup, and bathed in warm saline. A maintenance infusion of PBS/2.5% BSA was initiated at a rate of 0.2 μl/min per g body wt. After a 30-min equilibration period, micropuncture evaluation of TGF responses was performed as described previously (15). Proximal tubules were identified by injection of saline/0.25% Fast Green dye (Sigma, St. Louis, MO) into a random proximal segment. Early proximal portions were blocked with wax, and a micropipette attached to a nanoliter infusion pump was inserted into the last superficial proximal segment for loop of Henle perfusion with artificial tubule fluid (ATF; 136 mM NaCl, 4 mM NaHCO3, 4 mM KCl, 2 mM CaCl2, 7.5 mM urea, and 100 mg/ml Fast Green). For measurement of PSF, another micropipette attached to a servo-null pressure device (World Precision Instruments, Sarasota, FL), was inserted into an early proximal segment. Loop of Henle perfusion rate was altered from 0 to 40 nl/min, and TGF-mediated changes in PSF were recorded.

Protocols

Effect of Ouabain on TGF Responses in α1S/S/α2R/R and α1R/R/α2R/R Mice.

In the first series of experiments, TGF responses were determined in α1 ouabain-sensitive and -resistant mice before and during intravenous infusion of ouabain. After equilibration, a 30- to 45-min period during which one to three nephrons were identified for determination of control TGF responses was initiated. Animals then were given a bolus injection of ouabain at a dose of 65 ng/g body wt (in a volume of 2 μl/g body wt), followed by a constant infusion at a rate of 6.5 ng/g body wt per min. Assuming an extracellular fluid distribution for ouabain and clearance via glomerular filtration, it would be expected that this dosing regimen would result in an initial plasma concentration of approximately 0.5 μM and a gradually elevating concentration thereafter; a concentration of 0.1 to 1 μM is effective for inhibiting ouabain-sensitive Na,K-ATPase isoforms in a variety of biologic systems (16). After 20 min, TGF measurements were resumed and continued for up to 120 min. When the TGF response in any particular nephron seemed largely to be blocked (<20% of the control response), the perfusion pipette was exchanged for one that contained the adenosine agonist cyclohexyladenosine (CHA; 10 μM) to determine whether afferent arteriole responsiveness persisted in the presence of ouabain. Previous studies have demonstrated that intraluminal CHA can elicit strong TGF responses even when MD transport is blocked (17). In a second series of experiments, we tested the effect of intraluminal application of ouabain on TGF responses in α1S/S/α2R/R and α1R/R/α2R/R mice. After we determined the baseline TGF response in a nephron, we exchanged the perfusion pipette for one that contained 50 μM ouabain and reevaluated TGF responses at 0, 5, and 10 min. Between determinations, the loop of Henle was perfused with the ATF + ouabain at a slow rate (5 to 10 nl/min). When TGF responses were found to be blocked after intraluminal application of ouabain, the perfusion pipette was exchanged again for one that contained 50 μM ouabain and 10 μM CHA to determine whether responses could be observed. All measurements were made during a 2-h period after equilibration.

TGF Responses in NHE2 Knockout Mice and cHKA Knockout Mice.

Maximal TGF responses were determined in NHE2−/− and NHE2+/+ mice under normal conditions. Likewise, maximal TGF responses were determined in cHKA−/− and cHKA+/+ mice under control conditions.

Data Analysis

Arterial pressure and PSF recordings were analyzed using ADInstruments Chart software. Statistical analysis was performed by ANOVA using either a single-factor within-subjects design or a two-factor mixed design with repeated measures on the second factor. Individual contrasts were used to compare group means when needed. Data are presented as means ± SEM, and differences were regarded as significant at P < 0.05.

Results

BP data from all of the experiments are provided in Table 1, and values are given from the early, middle, and late portions of each experiment. For ouabain infusion experiments, this corresponds to midway through the control period and midway through the first and second hour of intravenous ouabain infusion. For all other experiments, values correspond to approximately the 20-, 60-, and 100-min time points. In the ouabain infusion protocol, mean arterial pressure in α1S/S/α2R/R mice increased immediately upon bolus injection of ouabain and remained elevated throughout the remainder of the experiment. In contrast, there was no response to infusion of ouabain in the α1R/R/α2R/R mice. Urine output in α1S/S/α2R/R mice increased from 1.5 ± 0.1 to 5.0 ± 0.9 μl/min in response to ouabain infusion (n = 5; P < 0.05). Urine flow rate did not change in response to ouabain in the α1R/R/α2R/R mice (1.2 ± 0.1 versus 1.3 ± 0.2 μl/min; n = 4). BP did not change over time in any of the remaining experiments. BP was not different between NHE2+/+ and NHE2−/− mice or between cHKA+/+ and cHKA−/− mice. We did not compare BP between the different mouse models because of differences in genetic background.

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Table 1.

BP in ouabain-sensitive and ouabain-resistant α1 Na,K-ATPase, NHE2 knockout and colonic H,K-ATPase knockout micea

TGF Responses in Na,K-ATPase Mutant Mice

In the ouabain infusion protocol, TGF responses in α1S/S/α2R/R mice were robust during the control period, with maximal changes in PSF averaging 9.7 ± 0.9 mmHg (Figure 1). Between 20 and 40 min and then 40 and 60 min of ouabain infusion, maximal TGF responses decreased to 6.2 ± 1.3 (P < 0.05 versus control) and 4.8 ± 2.1 mmHg (P < 0.02), respectively. Between 60 and 120 min of ouabain infusion, maximal TGF responses were nearly eliminated, with responses averaging only 1.6 ± 0.5 mmHg (P < 0.0001). In six nephrons in which responses had been eliminated by ouabain (indicated by dashed lines), robust decreases in PSF could be elicited by loop perfusion with 10 μM CHA, with maximal responses averaging 13.0 ± 2.7 mmHg (Figure 1, right). In α1R/R/α2R/R mice, intravenous administration of ouabain had no effect on TGF even after 2 h, as shown in Figure 2. Maximal responses were 14.7 ± 2.5 mmHg during the control period, 12.2 ± 1.1 after 20 to 40 min of ouabain, 15.0 ± 2.6 after 40 to 60 min of ouabain, and 16.3 ± 1.1 after 60 to 120 min of ouabain. We did not test CHA in these mice.

Figure 1.
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Figure 1.

Tubuloglomerular feedback (TGF) responses in α1-sensitive, α2-resistant mice (α1S/S/α2R/R; n = 7 mice; number of tubules as indicated) under control conditions (left) and at indicated times during intravenous infusion of ouabain (65 ng/g body wt bolus, followed by 6.5 ng/min per g body wt). Values for proximal stop-flow pressure (PSF) are shown at loop of Henle perfusion rate of 0 and 40 nl/min. Thin lines indicate responses in separate tubules. Thick lines are mean ± SEM. In some tubules (indicated by dotted lines), when blockade of the TGF response by ouabain appeared nearly complete, the ATF perfusion pipette was exchanged for one that contained artificial tubule fluid (ATF) + cyclohexyladenosine (CHA), and the response was repeated (right). P values for each PSF comparison (0 versus 40 nl/min) are as indicated. *P < 0.05 compared with the control response (ΔPSF).

Figure 2.
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Figure 2.

TGF responses in α1-resistant, α2-resistant mice (α1R/R/α2R/R; n = 4 mice; number of tubules as indicated) during control and intravenous ouabain infusion (see Figure 1 for details). Thin lines indicate responses in separate tubules. Thick lines are mean ± SEM. Because there was no apparent inhibition by ouabain, responses with CHA were not tested. P values for each comparison (0 versus 40 nl/min) are as indicated.

In the second protocol, we tested the effect of luminal ouabain on TGF, reasoning that in the area of the MD, the epithelium may be leaky enough to permit access to the basolateral surface from the luminal fluid. Results from α1S/S/α2R/R mice are shown in Figure 3, in which the TGF response is expressed as the maximal change (Δ) in PSF. During perfusion with ATF, control responses averaged 10.5 ± 1.1 mmHg, and after 10 min of loop perfusion with ATF + ouabain, responses had decreased to 3.9 ± 1.0 mmHg (P < 0.005). Further addition of 10 μM CHA to the loop perfusate produced constrictor responses even higher than those seen with ATF alone (19.9 ± 2.7 mmHg; P < 0.0005). These experiments were repeated in α1R/R/α2R/R mice, and no effect of luminal ouabain was observed, as shown in Figure 4.

Figure 3.
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Figure 3.

Maximal TGF responses in α1-sensitive, α2-resistant mice (α1S/S/α2R/R; n = 4 mice/8 tubules) while perfusing the downstream loop of Henle with ATF alone, ATF with ouabain (50 μM), and ATF with ouabain + CHA (10 μM). Values are expressed as the difference in PSF (ΔPSF) between loop perfusion rates of 0 and 40 nl/min. Ouabain values all were taken after 10 min of ouabain exposure. Ouabain + CHA were taken within 1 to 2 min of initial perfusion. Each set of measurements for one nephron is indicated by connected thin lines. Thick line represent means ± SEM. *P < 0.005 compared with response under control conditions.

Figure 4.
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Figure 4.

Maximal TGF responses in α1-resistant, α2-resistant mice (α1R/R/α2R/R; n = 4 mice/12 tubules) while perfusing the downstream loop of Henle with ATF alone and ATF with ouabain (50 μM). Values are expressed as the ΔPSF between loop perfusion rates of 0 and 40 nl/min. Ouabain values all were taken after 10 min of ouabain exposure. CHA was not tested because there was no consistent inhibition. Each set of measurements for one nephron is indicated by connected thin lines. Thick line represent means ± SEM.

TGF Responses in NHE2 and cHKA Knockout Mice

We evaluated TGF responses in NHE2+/+ and NHE2−/− mice, and results are shown in Figure 5. Maximal TGF responses were not different between the two groups, with changes in PSF averaging 14.4 ± 1.5 mmHg in the NHE2+/+ mice and 13.8 ± 0.8 mmHg in the NHE2−/− mice. Figure 6 shows the results from cHKA knockout mice. TGF responses were small in the cHKA−/− mice compared with other groups of animals evaluated in this study, with maximal PSF changes averaging 3.6 ± 0.7 mmHg. However, TGF responses in cHKA+/+ mice of the same genetic background also were small, and the PSF change of 3.7 ± 0.8 mmHg was not different from that in the cHKA−/− mice. Although it is not entirely clear why the TGF responses in these cohorts of mice was markedly lower than those seen in our other groups, it may be related to the low BP achieved under anesthesia in these mice (see Table 1). In support of this notion, PSF under conditions of zero loop flow rate also was lower in these animals than in other groups (approximately 30 mmHg). It is interesting that in the one cHKA−/− tubule with a PSF near 40 mmHg, the TGF response was robust (Figure 6, right).

Figure 5.
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Figure 5.

TGF responses in NHE2 knockout (NHE2−/−; n = 5 mice/19 tubules) and wild-type mice (NHE2+/+; n = 4 mice/15 tubules) under euvolemia. Thin lines indicate responses in individual tubules. Thick lines are mean ± SEM. Responses were not different between genotypes. *P < 0.0001 compared with 0 nl/min flow.

Figure 6.
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Figure 6.

TGF responses in colonic H,K-ATPase knockout (cHKA−/−; n = 4 mice/13 tubules) and wild-type mice (cHKA+/+; n = 5 mice/12 tubules) under euvolemia. Thin lines indicate responses in individual tubules. Thick lines are mean ± SEM. Responses were not different between genotypes. *P < 0.001 compared with 0 nl/min flow.

Discussion

Several findings in this study enhance our understanding of the transport events in the juxtaglomerular apparatus (JGA) that participate in TGF signaling. First, because both intravenous and intraluminal ouabain inhibited TGF responses in the α1S/S/α2R/R mice but not in the α1R/R/α2R/R mice, our data provide functional evidence that the α1 isoform of the Na,K-ATPase plays a central role in TGF signaling. Because application of the adenosine analogue CHA to the lumen produced robust decreases in PSF, the inhibitory effect of ouabain was not due to a general inability of the afferent arteriole to constrict to appropriate stimuli but more likely to inhibition of α1 Na,K-ATPase in MD cells. Finally, our data from NHE2 and cHKA knockout mice establish that neither of these transport pathways is essential for the generation of TGF signals. It is important to note, however, that the persistence of TGF responsiveness in the NHE2 and cHKA knockout mice does not preclude the possibility that these transporters may importantly influence the sensitivity of the TGF signaling cascade.

Intravenous administration of ouabain had a marked effect on both BP and overall renal function as evidenced by more than a three-fold increase in urine flow rate. Although BP can have an important influence on the magnitude of TGF responsiveness, it is well established that increases in pressure are associated with enhanced TGF responses (18). It therefore is unlikely that the observed inhibition of TGF was due to an indirect effect of the BP elevation.

Recent studies by Bell and Peti-Peterdi and colleagues have elucidated the existence and participation of several important ion transport pathways in MD-generated signals. Using functional analysis of intracellular pH and Na+ concentration in the isolated perfused JGA from rabbit, they demonstrated the existence of Na+/H+ exchangers on both the apical (NHE2) and basolateral (NHE4) membranes of MD cells and provided compelling evidence that MD [Na+]i directly correlates with luminal NaCl concentration in a manner that parallels the well-described TGF response (6). They further demonstrated that these changes in MD [Na+]i were sensitive to luminal but not bath application of ouabain and that there was a K-dependent alkalinization of MD cells that could be blocked by ouabain (but not the gastric H,K-ATPase inhibitor Sch-28080). Because an earlier immunohistochemical study had already demonstrated MD expression of the H,K-ATPase α2c subunit, a splice variant of the rabbit colonic HKA (7), it was concluded that apical expression of cHKA, acting as a Na(H),K-ATPase and/or in conjunction with apical Na/H exchange, is responsible for regulating MD cytosolic Na+ concentration (6). With the caveat that these earlier studies were performed in rabbit, our data in knockout mice showing that neither NHE2 nor cHKA is required for the generation of TGF signal argues against a central role for these transporters in maintaining the necessary electrochemical gradient for activity of the Na-K-2Cl-co-transporter and by inference the initiation of the TGF signaling pathway. In contrast, the ability of ouabain to inhibit TGF responses in α1-sensitive but not α1-resistant mice supports the hypothesis that MD Na,K-ATPase activity is an integral component in the transmission of the TGF signal.

These findings regarding a central role for MD Na,K-ATPase activity in the generation of MD signaling does not rule out an important role for apical transporters in modulating these signals. Although our data clearly demonstrate the presence of intact TGF in the NHE2 and cHKA knockout mice, we cannot comment on the ability of these apical transport pathways to alter the magnitude or the sensitivity of the TGF response. In this regard, several studies have proposed an important role for intracellular pH in altering TGF responsiveness. Bell and colleagues (19) found that MD cytosolic pH increased in response to elevated luminal Na+ concentration and that this response could be blunted by inhibition of Na/H exchange with amiloride or augmented by treatment with angiotensin II (20). Indeed, in a recent series of studies, Garvin and colleagues (21) reported that TGF responses could be enhanced by amiloride and by inhibition of neuronal nitric oxide (NO) synthase. It was concluded that changes in cytosolic pH that accompany the initiation of TGF result in an increased NO production that in turn serves to limit the magnitude of the TGF response. Recent experiments that directly measured MD NO production support this hypothesis (22). In our NHE2 knockout mice, TGF responses, although large, were not different from control. However, a more thorough analysis under varying conditions such as volume loading or treatment with angiotensin II is required. It may be that the proposed pH-dependent mechanism has more to do with long-term resetting of the TGF sensitivity than with the actual moment to moment signaling cascade.

The use of ouabain alone as a pharmacologic tool to dissect the activity and the role of various ATPases is precarious. As already mentioned, the sensitivity of the Na,K-ATPase varies between isoforms and, to an even greater degree, between species. Furthermore, the ouabain sensitivities of the various H,K-ATPases are even more uncertain. For example, the sensitivity (EC50) of renal H,K-ATPase to ouabain has been reported to range from approximately 1 mM in a Xenopus oocyte cHKA expression system to 20 μM for the type III K-ATPase in the in rat collecting duct (23). Given this level of uncertainty, it is difficult to draw conclusions on the basis of the effects of high doses of ouabain in most experimental systems. In this study, however, the contrasting effects of ouabain in α1S/S/α2R/R mice versus α1R/R/α2R/R mice strengthens the conclusion that the α1 Na,K-ATPase is the target for the ouabain-induced inhibition of TGF, because the specific mutation of the α1 N,K-ATPase is the only difference between the two groups of mice.

In our second series of experiments, we administered ouabain to the JGA via the luminal perfusate, anticipating that ouabain may be able to gain access to the basolateral membrane by crossing the tubular epithelium (hence our choice of a moderately high dosage of 50 μM intraluminal ouabain). Previous studies suggested that the MD cell plaque is permeable to water (24), and the well-demonstrated ability of luminal CHA to produce afferent arteriolar constriction in this and other studies (17) suggests that it is not unreasonable that molecules in this size range (molecular weight of CHA is 349 versus 585 for ouabain) might gain access to the basolateral surface from the tubular lumen. Because ouabain did indeed block TGF responses in α1S/S/α2R/R mice but not in α1R/R/α2R/R mice and because it is highly unlikely that the α1 Na,K-ATPase is expressed on the apical membrane, it is only reasonable to conclude that ouabain did indeed exert its effect at the basolateral surface. In terms of the earlier studies of Peti-Peterdi et al. (6) using the isolated perfused JGA, they observed that administration of 1 mM ouabain to the lumen but not bath inhibited MD [Na+]i responses to changes in luminal NaCl. Although this initially may suggest an apical site of action, it is possible that the glomerulus and extramesangial cell cushion that overlays the MD cell plaque in the isolated perfused preparation represents a more formidable barrier for access to the basolateral membrane than does the single layer of epithelial cells. Given the high concentrations of ouabain administered to the lumen in those studies (1 mM versus 50 μM in our study) and the high sensitivity of the rabbit α1 Na,K-ATPase to ouabain, their results might be explained by the diffusion of ouabain from the lumen to the basolateral membrane.

Compared with baseline TGF responses in other groups of mice, which average 10 to 15 mmHg, the responses in cHKA knockout and wild-type mice were relatively small, averaging only 3.5 to 4 mmHg. We suspect that this likely reflects the lower BP exhibited under anesthesia by the pure C57BL/6 genetic background on which this colony was maintained, falling below a critical autoregulatory threshold of 85 to 90 mmHg (25). In any case, the data must be regarded with some caution. We are confident only in our conclusion that TGF responses can be elicited in the absence of cHKA but cannot speculate as to whether the magnitude of these responses might be altered under more physiologic conditions. Additional studies using cHKA-deficient mice that have been backcrossed onto a more robust background will be necessary to address this question fully.

Conclusion

The data presented here provide evidence that the α1 isoform of the Na,K-ATPase participates in the transmission of the MD signal for TGF. Because strong afferent arteriolar constriction could be elicited by the adenosine analogue CHA after TGF responsiveness had been blocked by ouabain, the data are consistent with the hypothesis that even the relatively low level of Na,K-ATPase expressed in MD cells is integral in establishing the inward Na+ gradient that energizes other transport processes. Finally, the presence of TGF responses in NHE2 and cHKA knockout mice suggests that these apical pathways are not essential for establishing the presence of TGF but does not rule out the possibility that they may participate in modulating the sensitivity of the response. In light of the heightened interest in the role of endogenous ouabain-like substances in modulating the activity of the Na,K-ATPases (26,27), these findings may reflect an as-yet-undefined but potentially important regulatory pathway for juxtaglomerular signaling and control of renal hemodynamics.

Acknowledgments

This work was supported by National Institutes of Health grants DK57552 (to J.N.L.), HL66062 and HL 28573 (to J.B.L.), and DK 50594 (to G.E.S.).

We thank J. Schnermann and R. Banks for thoughtful review of the manuscript.

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

  • © 2006 American Society of Nephrology

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Journal of the American Society of Nephrology: 17 (9)
Journal of the American Society of Nephrology
Vol. 17, Issue 9
September 2006
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Ouabain Inhibits Tubuloglomerular Feedback in Mutant Mice with Ouabain-Sensitive α1 Na,K-ATPase
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Ouabain Inhibits Tubuloglomerular Feedback in Mutant Mice with Ouabain-Sensitive α1 Na,K-ATPase
John N. Lorenz, Iva Dostanic-Larson, Gary E. Shull, Jerry B. Lingrel
JASN Sep 2006, 17 (9) 2457-2463; DOI: 10.1681/ASN.2006040379

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Ouabain Inhibits Tubuloglomerular Feedback in Mutant Mice with Ouabain-Sensitive α1 Na,K-ATPase
John N. Lorenz, Iva Dostanic-Larson, Gary E. Shull, Jerry B. Lingrel
JASN Sep 2006, 17 (9) 2457-2463; DOI: 10.1681/ASN.2006040379
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More in this TOC Section

  • Role of Microsomal Prostaglandin E Synthase 1 in the Kidney
  • Pituitary Adenylate Cyclase–Activating Polypeptide Stimulates Renin Secretion via Activation of PAC1 Receptors
  • Lack of Endothelial Nitric Oxide Synthase Promotes Endothelin-Induced Hypertension: Lessons from Endothelin-1 Transgenic/Endothelial Nitric Oxide Synthase Knockout Mice
Show more Hemodynamics and Vascular Regulation

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