Ouabain Inhibits Tubuloglomerular Feedback in Mutant Mice with Ouabain-Sensitive α1 Na,K-ATPase

Materials and Methods

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 α1^S/S/α2^R/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 α1^R/R/α2^R/R mice. Urine output in α1^S/S/α2^R/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 α1^R/R/α2^R/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.

TGF Responses in Na,K-ATPase Mutant Mice

In the ouabain infusion protocol, TGF responses in α1^S/S/α2^R/R mice were robust during the control period, with maximal changes in P_SF 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 P_SF could be elicited by loop perfusion with 10 μM CHA, with maximal responses averaging 13.0 ± 2.7 mmHg (Figure 1, right). In α1^R/R/α2^R/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.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Tubuloglomerular feedback (TGF) responses in α1-sensitive, α2-resistant mice (α1^S/S/α2^R/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 (P_SF) 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 P_SF comparison (0 versus 40 nl/min) are as indicated. *P < 0.05 compared with the control response (ΔP_SF).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

TGF responses in α1-resistant, α2-resistant mice (α1^R/R/α2^R/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 α1^S/S/α2^R/R mice are shown in Figure 3, in which the TGF response is expressed as the maximal change (Δ) in P_SF. 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 α1^R/R/α2^R/R mice, and no effect of luminal ouabain was observed, as shown in Figure 4.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

Maximal TGF responses in α1-sensitive, α2-resistant mice (α1^S/S/α2^R/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 P_SF (ΔP_SF) 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.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4.

Maximal TGF responses in α1-resistant, α2-resistant mice (α1^R/R/α2^R/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 ΔP_SF 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 P_SF 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 P_SF 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, P_SF 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 P_SF near 40 mmHg, the TGF response was robust (Figure 6, right).

• Download figure
• Open in new tab
• Download powerpoint

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.

• Download figure
• Open in new tab
• Download powerpoint

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 α1^S/S/α2^R/R mice but not in the α1^R/R/α2^R/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 P_SF, 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 α1^S/S/α2^R/R mice versus α1^R/R/α2^R/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 α1^S/S/α2^R/R mice but not in α1^R/R/α2^R/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.