Ammonia production and secretion by S3 proximal tubule segments from acidotic mice: role of ANG II

【摘要】  ANG II has potent effects on ammonia production and secretion rates by the proximal tubule and is found in substantial concentrations in the lumen of the proximal tubule in vivo. Because our previous studies demonstrated that acid loading enhanced the stimulatory effects of ANG II on ammonia production and secretion by S2 proximal tubule segments, we examined the effect of ANG II on ammonia production and secretion by isolated, perfused S3 segments from nonacidotic control mice and acidotic mice given NH 4 Cl for 7 days. In the absence of ANG II, ammonia production and secretion rates were no different in S3 segments from acidotic and control mice. By contrast, when ANG II was present in the luminal perfusion solution, ammonia production and secretion rates were stimulated, in a losartan-inhibitable manner, to a greater extent in S3 segments from acidotic mice. Ammonia secretion rates in S3 segments were largely inhibited by perfusion with a low-sodium solution containing amiloride in the presence or absence of ANG II. These results demonstrated that isolated, perfused mouse S3 proximal tubule segments produce and secrete ammonia, that NH 4 Cl-induced acidosis does not affect the basal rates of ammonia production and secretion, and that ANG II, added to the luminal fluid, stimulates ammonia production and secretion to a greater extent in S3 segments from acidotic mice. These findings suggest that S3 segments, in the presence of ANG II, can contribute to the enhanced renal excretion that occurs with acid loading.

【关键词】  ammonia transport ammoniagenesis acidbase physiology losartan

THE PROXIMAL TUBULE IS THE major site of regulation of total ammonia (tNH 3 = NH 3 + NH 4 + ) production in the kidney, and the tNH 3 that reaches the late portions of the proximal tubule accounts for most of the tNH 3 that appears in the final urine ( 8, 11 ). With acid loading, tNH 3 production and secretion rates in the proximal tubule undergo adaptive increases that are largely the result of changes in transport and enzymatic activities in the proximal tubule ( 7, 18, 27, 29 ). From previous studies, the adaptive increase in tNH 3 production rate in response to acid loading appeared to be limited to the early segments of the proximal tubule (S1 and S2; see Refs. 9, 24, 25 ). The late straight portion (S3) segment of the proximal tubule did not display an adaptive increase in tNH 3 production rates after acid loading ( 9 ).

Our previous studies demonstrated that ANG II plays an important role in regulating tNH 3 production and secretion by the S2 proximal tubule segment ( 16, 17 ) and is critically important for the adaptive increase in tNH 3 secretion that is observed with short-term acid loading ( 19 ). Furthermore, ANG II at certain concentrations stimulates tNH 3 production by the S2 proximal tubule segment, and this stimulatory effect is heightened by acid loading in vivo ( 19 ). The physiological importance of ANG II on tubular function is further underscored by studies indicating that ANG II is present in the lumen of proximal tubule in concentrations that are similar to those that stimulate tNH 3 production and secretion ( 4, 23, 28 ). Previous studies on the effects of acidosis on tNH 3 production rates by the proximal tubule segments were performed in unperfused, dissected nephron segments in the absence of ANG II ( 9, 24, 25 ). These findings formed the basis for reexamining the effects of acidosis on tNH 3 production and secretion by isolated, perfused mouse S3 segments in the presence or absence of ANG II.

In the present study, we examined the rates of tNH 3 production and secretion by isolated, perfused S3 proximal tubule segments from acidotic and nonacidotic mice, measured their response to ANG II, and ascertained whether acid loading in vivo resulted in a difference in the stimulatory effects of ANG II on tNH 3 production and secretion rates. Our data indicate that the S3 segment produces tNH 3 in substantial amounts, that a substantial portion of the tNH 3 that is produced in S3 segment cells is secreted in the tubule lumen, that addition of ANG II to the luminal perfusion solution markedly enhances tNH 3 secretion and production rates, and that acid loading significantly increased the stimulatory effects of ANG II on the rates of tNH 3 production and secretion. These studies suggest that acidosis induces changes in tNH 3 production and secretion in the S3 proximal tubule segment that may become evident in the presence of ANG II.

MATERIALS AND METHODS

Animals. The studies performed were approved by the Animal Research Committee at the Veterans Affairs Greater Los Angeles Healthcare System. Male Swiss-Webster mice (Hilltop, Scottdale, PA) weighing 25-30 g were maintained on Purina Rodent Chow. Mice were provided 0.3 M NH 4 Cl in 2% sucrose in water or 2% sucrose alone in water. At the end of 7 days, the mice were anesthetized with intramuscular injections of ketamine (0.09 mg/g body wt) and xylazine (0.01 mg/g body wt), and blood was obtained from the aorta for measurement of plasma total CO 2. Urine was obtained from the bladder for determination of tNH 3 and creatinine.

Microperfusion of mouse proximal tubule segments. Outer cortical proximal tubules were identified under direct microscopic visualization, and S3 segments of the mouse proximal tubule comprising the late straight portions (0.8 ± 0.2 mm) were obtained by using needle-tipped forceps to dissect down the proximal tubule to the straight portion in kidney slices bathed in precooled buffer. An S3 segment was placed in a temperature-regulated chamber mounted over the objective of an inverted microscope and was microperfused using concentric pipettes so that the luminal aspect of the S3 segment was cannulated and perfused with Krebs-Ringer bicarbonate (KRB) buffer. The flow rates (20.1 ± 0.3 nl/min) did not differ among the groups studied. The S3 segment was bathed in 300 µl KRB buffer containing 0.5 mM L -glutamine pregassed with 95% O 2 -5% CO 2, pH 7.4 at 37°C.

Measurement of luminal tNH 3 secretion rates. Net luminal tNH 3 secretion rates were measured by collecting the fluid leaving the distal end of the perfused segment with a pipette over a 20-min period ( 15, 19 ). The tNH 3 content in the collected fluid was measured using a microenzymatic method coupling the conversion of 2-oxoglutarate, NADH, and tNH 3 to NAD + and glutamate ( 15, 21 ). Luminal tNH 3 secretion rates equaled the rate at which tNH 3 left the distal end of the perfused segment in timed luminal fluid collections.

Measurement of tNH 3 production rates. The rates of tNH 3 production by isolated, perfused S3 segments were determined by adding the rates of luminal secretion with the rates of accumulation of tNH 3 in the bath solution, which was covered with pregassed mineral oil and continuously bubbled with a gas jet of 95% O 2 -5% CO 2. At the end of a 20-min incubation period, an aliquot of the bath solution was taken for analysis of tNH 3. The volume of the bath solution was determined from the degree of dilution of Trypan blue dye added in known amounts to the bath solution at the completion of the study.

Measurements of total CO 2 concentrations. Total CO 2 was determined on serum samples enzymatically using the phospho enol pyruvate carboxykinase reaction ( 19 ). The measurements were linear over the range of concentrations observed.

Solutions. KRB buffer solution contained the following electrolytes (in mM): 125 NaCl, 25 NaHCO 3, 5 KCl, 1 MgCl 2, 1 NaH 2 PO 3, and 1 CaCl 2. ANG II and losartan (Merck) were used in concentrations as specified in RESULTS. The low-sodium + amiloride perfusion solution substituted N -methyl glucamine chloride for NaCl and partially substituted choline bicarbonate for sodium bicarbonate in the KRB buffer solution (sodium concentration 10 mM), and amiloride was added at a final concentration of 0.1 mM. In specified experiments, the pH of luminal perfusion solution was lowered by lowering the bicarbonate concentration. All glutamine-containing solutions were freshly prepared using the purest form of L -glutamine available (Sigma Chemical).

Statistical analysis. Comparisons between two groups of data were done using Student's t -test, whereas comparisons among multiple groups were made using ANOVA with multiple comparisons by the method of Scheffé ( 26 ). All data are presented as means ± SE.

RESULTS

Effect of 7 days NH 4 Cl loading on serum total CO 2 and urinary tNH 3 excretion. Mice given NH 4 Cl + sucrose in their drinking water for 7 days developed a reduction in serum bicarbonate (total CO 2 ) and increased rates of urinary tNH 3 excretion ( Table 1 ). These results were similar to those observed by us in mice previously and are consistent with the development of a metabolic acidosis ( 22 ).

Table 1. Effect of NH 4 Cl loading for 7 days on bicarbonate concentration and urinary tNH 3 excretion rates

Effect of ANG II on tNH 3 production by microperfused S3 segments. We examined the effects of a range concentrations of ANG II added to the luminal perfusion solution on tNH 3 production rates in S3 proximal tubule segments. As shown in Fig. 1, luminal ANG II had concentration-dependent effects on tNH 3 production rates by microperfused S3 segments. Addition of 10 -11 and 10 -6 M ANG II to the luminal perfusion solution resulted in tNH 3 production rates that were not significantly different from rates observed in segments perfused in the absence of ANG II [ANG II 10 -11 ( n = 5): 12.5 ± 1.1 pmol·min -1 ·mm -1; ANG II 10 -6 M ( n = 5): 10.0 ± 0.9; control ( N = 5): 11.5 ± 0.9]. On the other hand, addition of 10 -10 or 10 -9 M ANG II to the luminal fluid significantly stimulated tNH 3 production rates [ANG II 10 -10 ( n = 5): 31.0 ± 1.0; ANG II 10 -9 ( n = 5): 34.4 ± 1.7 pmol·min -1 ·mm -1, P < 0.05].

Fig. 1. ANG II had concentration-dependent effects on total ammonia production rates by S3 proximal tubule segments from control mice (* P < 0.05 vs. other groups).

Effect of acidosis on tNH 3 production and secretion rates in microperfused S3 segments from control and acidotic mice. The rates of tNH 3 production in S3 proximal tubule segments were measured in segments derived from control and acidotic mice ( Fig. 2, conditions A and E ). In the absence of ANG II, S3 segments derived from control and acidotic mice produced tNH 3 at rates that were not significantly different from each other (11.5 ± 0.9 vs. 12.9 ± 1.1 pmol·min -1 ·mm -1, n = 5). As shown in Fig. 2 ( conditions A and E ), the rates of secretion of tNH 3 in the tubule lumen also did not significantly differ in S3 segments from control and acidotic mice (6.5 ± 0.6 vs. 6.8 ± 0.5 pmol·min -1 ·mm -1, n = 5) in the absence of ANG II. The percentage of the tNH 3 produced that was secreted in the lumen in the S3 segment was similar in segments from control and acidotic mice (56 vs. 53%, respectively). Thus, in the absence of ANG II, acidosis did not induce an adaptive increase in tNH 3 production or secretion rates.

Fig. 2. Total ammonia production rates by S3 proximal tubule segments from control (open bars) and acidotic (gray bars) mice in the presence or absence of 10 -9 M ANG II or 10 -5 M losartan. In the absence of losartan, luminal ANG II resulted in higher total ammonia production rates in S3 segments from control mice ( condition C vs. A ) and resulted in still higher rates in S3 segments from acidotic mice ( conditions G vs. all other groups). Losartan blocked the stimulatory effect of ANG II (* P < 0.05 compared with all other groups).

Effect of ANG II on tNH 3 production rates by microperfused S3 segments from control and acidotic mice. As shown in Fig. 2, the addition of 10 -9 M ANG II to the luminal perfusion solution resulted in higher rates of tNH 3 production by S3 segments from control mice ( condition C : 34.4 ± 1.7, n = 5, P < 0.01) and from acidotic mice ( condition G : 43.5 ± 1.3, n = 5, P < 0.01). The rates of tNH 3 production by S3 segments from acidotic mice were higher than those by segments from nonacidotic controls ( P < 0.05). As shown in Fig. 2, the stimulatory effect of ANG II on tNH 3 production rates was inhibited by addition of losartan, a type 1 ANG II (AT 1 ) receptor blocker, to the luminal perfusion solution along with ANG II. Losartan reduced the tNH 3 production rates observed with exposure to luminal ANG II in S3 segments from control mice ( condition D : 13.6 ± 1.2 pmol·min -1 ·mm -1, n = 5) and in segments from acidotic mice ( condition H : 14.1 ± 0.9, n = 5). Addition of losartan by itself had no significant effect on production rates by S3 segments from control (11.9 ± 0.9 pmol·min -1 ·mm -1, n = 5) and acidotic (12.5 ± 1.1, n = 5) mice.

Effect of ANG II on tNH 3 secretion rates in microperfused S3 segments from control and acidotic mice. As depicted in Fig. 3, ANG II stimulated tNH 3 secretion rates in S3 segments from nonacidotic and acidotic mice. The rate of net tNH 3 secretion in the luminal fluid was higher when S3 segments from nonacidotic mice were perfused with 10 -9 M ANG II ( condition C : 14.4 ± 0.9 pmol·min -1 ·mm -1, n = 5, P < 0.01 compared with condition A ) and was even higher in S3 segments from acidotic mice ( condition G : 22.5 ± 1.6, n = 5, P < 0.05 compared with all other conditions). Although the absolute rates of tNH 3 secretion were higher in ANG II-stimulated S3 segments from acidotic mice compared with those from control mice, the percentage of the tNH 3 produced that was secreted in the lumen was 41% in S3 segments from control mice and 52% in those from acidotic mice. Losartan blocked the stimulatory effect of ANG II on net luminal tNH 3 secretion rates. Thus ANG II added to the luminal perfusion solution markedly stimulated tNH 3 production and secretion rates by S3 proximal tubule segments, and the stimulatory effect was higher in S3 segments derived from acidotic mice.

Fig. 3. Rates of luminal total ammonia secretion rates by S3 proximal tubule segments from control (open bars) and acidotic (gray bars) mice in the presence or absence of 10 -9 M ANG II or 10 -5 M losartan. In the absence of losartan, luminal ANG II resulted in higher luminal total ammonia secretion rates in S3 segments from control mice ( condition C vs. A ) and resulted in still higher rates in S3 segments from acidotic mice ( conditions G vs. all other groups). Losartan blocked the stimulatory effects of ANG II in S3 segments from control and acidotic mice (* P < 0.05 compared with all other groups).

Effect of luminal perfusion with amiloride in a low-sodium modified KRB solution on net luminal tNH 3 secretion in S3 segments. As in previous studies on S2 segments ( 19 ), we examined the impact of reducing the sodium concentration (to 10 mM) in and adding amiloride (0.1 mM) to the luminal perfusion solution on net tNH 3 secretion by S3 proximal tubule segments. In some experiments, the pH of the low-sodium + amiloride luminal perfusion solution was lowered to 6.2 by lowering the bicarbonate concentration in the perfusion solution to 1.5 mM. Perfusion with the amiloride-containing low-sodium perfusion solution inhibited net luminal tNH 3 secretion rates ( Fig. 4 ). In the absence of luminal ANG II, perfusion with the amiloride-containing low-sodium perfusion solution significantly reduced net luminal tNH 3 secretion (control 6.7 ± 1.0 pmol·min -1 ·mm -1 vs. amiloride + low sodium 1.2 ± 1 pmol·min -1 ·mm -1, n = 5, P < 0.05). In the presence of ANG II, perfusion with the amiloride-containing solution inhibited net tNH 3 secretion in control S3 segments (+ANG II 15.0 ± 1.3 vs. +ANG II + amiloride + low sodium: 2.1 ± 0.9 pmol·min -1 ·mm -1, n = 5, P < 0.01) and in S3 segments from acidotic mice (+ANG II: 22.2 ± 1.5 pmol·min -1 ·mm -1 vs. +ANG II + amiloride + low sodium: 2.0 ± 1.1, n = 5, P < 0.01). Perfusion with 0.1 mM amiloride in standard KRB buffer did not significantly affect net luminal tNH 3 secretion rates in S3 segments from control (7.0 ± 1.0 pmol·min -1 ·mm -1, n = 5) or acidotic (6.9 ± 0.9 pmol·min -1 ·mm -1, n = 5) mice. In other words, an inhibitory effect of 0.1 mM amiloride did not occur without the reduction in luminal sodium concentration, suggesting relative resistance to the effects of amiloride. Perfusion with the low-sodium solution containing amiloride did not affect tNH 3 production rates in the presence or absence of ANG II (low sodium + amiloride without ANG II: 12.1 ± 1.0; low sodium + amiloride with ANG II: 33.9 ± 2.0 pmol·min -1 ·mm -1 ) compared with rates observed in S3 segments from control mice, indicating that a major portion of the tNH 3 produced by the proximal tubule cells was released in the bath solution rather than in the luminal perfusion solution when the S3 segments were perfused with the low-sodium + amiloride perfusion solution.

Fig. 4. Addition of amiloride (Amil) to a low-sodium perfusion solution markedly inhibited the ANG II-stimulated net luminal tNH 3 secretion rates observed in S3 segments from control mice (open bars) and acidotic mice (gray bars) in the presence or absence of ANG II. Lowering the perfusion solution pH to 6.2 did not restore the tNH 3 secretion rates to those observed with ANG II using the control luminal Krebs-Ringer bicarbonate perfusion solution (* P < 0.05 vs. all other groups; P < 0.05 compared with the group perfused with normal KRB in the absence of ANG II).

Results of additional studies shown in Fig. 4 demonstrated the impact of lowering the luminal pH of the low-sodium solution containing amiloride on net luminal tNH 3 secretion. Lowering the pH of the perfusion solution to 6.2 in the presence of ANG II in the luminal low-sodium + amiloride perfusion solution resulted in only a partial restoration of the ANG II-stimulated net luminal tNH 3 secretion rates in S3 proximal tubule segments from control mice (6.2 ± 0.9 pmol·min -1 ·mm -1, n = 5) or from acidotic mice (9.2 ± 1.0 pmol·min -1 ·mm -1, n = 5). Thus lowering the luminal pH by markedly lowering the luminal perfusion solution pH in the presence of amiloride and a low-sodium concentration resulted in an incomplete restoration of tNH 3 secretion rates observed with perfusion with the control KRB perfusion solution containing ANG II. These results suggest that the mechanisms involved in luminal tNH 3 secretion by isolated, perfused S3 proximal tubule segments are inhibited by conditions that may inhibit Na + /H + (NH 4 + ) exchanger activity and that acidification of the luminal fluid by lowering the pH of the low-sodium + amiloride perfusion solution did not completely restore tNH 3 secretion rates.

DISCUSSION

The production and secretion of tNH 3 by the proximal tubule are critically important in the way that the kidney defends against acid challenges ( 18 ). Although previous studies have suggested that S3 proximal tubule segments contribute little to the adaptive enhancement of tNH 3 excretion by the kidney observed with acid loading, the present study demonstrated that, in the presence of ANG II in the luminal fluid, the S3 segment from acidotic mice displays significantly enhanced rates of tNH 3 production and secretion. Thus adaptive enhancements in tNH 3 secretion and production rates by the S3 segment acidosis became apparent in the presence of ANG II.

Studies on unperfused rat proximal tubule segments by Good and Burg ( 9 ) demonstrated that S3 segments from acidotic rats do not produce tNH 3 at a higher rate than those from nonacidotic control rats, whereas S1 and S2 segments from acidotic rats produced tNH 3 at significantly higher rates than those from control rats. In the present study, microperfused S3 segments from acidotic mice and nonacidotic control mice showed no difference in rates of tNH 3 production or secretion in the absence of ANG II. Thus S3 segments did not display an adaptive increase in either tNH 3 production or secretion rates. This observation suggested that perfusion of S3 segments did not unmask an adaptive change not evident in unperfused S3 segments.

The apparent failure of the S3 segment to enhance its tNH 3 production rates with acidosis has been explained by a lack of induction of key ammoniagenic enzymes in response to acidosis. Studies by others have demonstrated that certain key enzymes of ammoniagenesis, including glutamate dehydrogenase and phosphate-dependent glutaminase, are present in all segments of the proximal tubule but increase with chronic metabolic acidosis in only the early portions of the proximal tubule (S1 and S2 segments) and not in the more distal S3 segment ( 6, 30, 31 ). Thus the machinery for producing tNH 3 is present in all segments of the proximal tubule, but adaptive enhancement of the expression of key ammoniagenic enzymes appears limited to the early segments of the proximal tubule.

The absence of an adaptive rise in luminal tNH 3 secretion rates in the S3 segment could be explained by a lack of enhancement in the apical Na + /H + exchanger expression in this segment. The major apical Na + /H + exchanger, the NHE3 isoform, in the proximal tubule can serve as an Na + /NH 4 + exchanger ( 12, 15 ). It is unclear whether NHE3 is expressed in all segments of the proximal tubule ( 2, 3 ). NHE3 expression is enhanced by acidosis in the S1 and S2 segments of the proximal tubule ( 1 ), but enhancement was not reported in the S3 segment. Thus differences in NHE3 expression along the proximal tubule or in response to acid loading may explain the differences in the observed effects of acid loading on tNH 3 secretion rates in mouse S2 proximal tubule segments ( 19 ) and mouse S3 segments, as observed in the present study.

The results of the present studies demonstrate that adaptive increases in tNH 3 production and secretion rates with chronic acid loading may be observed in S3 segments when the rates are measured in S3 segments exposed to luminal ANG II. ANG II plays an important role in the regulation of tNH 3 production and transport ( 5, 16, 17, 19 ), and ANG II is present in the fluid of the proximal tubule lumen in substantial concentrations ( 4, 23, 28 ). In the S2 proximal tubule segment, ANG II can stimulate rates of tNH 3 production and secretion to a greater extent in segments from acid-loaded mice compared with those from controls ( 19 ). As in the S2 proximal tubule segment, ANG II enhanced the rates of tNH 3 production and secretion by S3 proximal tubule segments in a manner that was inhibited by the AT 1 receptor blocker losartan. Furthermore, the stimulatory effects of ANG II on tNH 3 production and secretion were greater in S3 segments obtained from acidotic mice compared with those from control mice. Acute addition of ANG II to the luminal fluid stimulates, within minutes, tNH 3 production rates before new enzymes can be induced. The stimulation of ammoniagenesis by acute stimuli may be because of enhanced fluxes of metabolites through key ammoniagenic enzyme pathways ( 13 ). The mechanism for the enhancement of the stimulatory effect of ANG II on tNH 3 production rates is not known, but enhanced receptor expression with acid challenges has been observed in preliminary studies on cultured proximal tubule cells ( 20 ). Thus S3 proximal tubule segments from acidotic mice respond to ANG II via AT 1 receptors with greater enhancement in tNH 3 production and secretion rates, thereby increasing the contribution of this segment to the delivery of tNH 3 in the luminal fluid by this segment.

Although in some ways the secretion of tNH 3 by S3 segments is similar to that observed by S2 segments, the mechanisms of secretion of tNH 3 in the two segments may differ. In our previous studies using mouse S2 proximal tubule segments, we demonstrated that most (50-60%) of the tNH 3 produced by this proximal tubule segment is secreted in the luminal fluid despite the more favorable diffusion gradient allowing tNH 3 to escape to the surrounding bath solution ( 15 ) and that addition of ANG II to the luminal perfusion solution markedly increased the fraction of tNH 3 that was secreted in the lumen relative to the rate of production ( 80%). The results from the present study indicate that the fraction of tNH 3 that is secreted for a given amount produced by the S3 segment is 56%, similar to the fraction observed in S2 segments. However, with addition of ANG II to the luminal fluid, although the absolute rate of tNH 3 secretion was increased markedly, the fraction of the tNH 3 produced that was secreted in the lumen fell to 42%. These results suggested differences between the ANG II-stimulated tNH 3 secretory mechanisms in the mouse S2 and S3 proximal tubule segments. In S3 segments, enhanced rates of ammoniagenesis, by providing a larger transportable intracellular pool of tNH 3, may be responsible for the enhancement in tNH 3 secretion rather than direct stimulation of secretory mechanisms, which may be more likely the case in the S2 segment.

Studies in rabbit S3 segments suggested that, when tNH 3 was present in equal concentrations on the basolateral and luminal aspects of the tubule segment, the concentration of tNH 3 in the lumen was enhanced by the creation of an acid disequilibrium pH, suggesting a role for diffusion of nonionic NH 3 and trapping of NH 4 + ( 14 ). Our previous studies in S2 segments suggested that net luminal tNH 3 secretion was mediated by an apical Na + /H + exchanger acting as an Na + /NH 4 + exchanger and that an artificially reduced luminal pH in the absence of Na + /H + exchanger activity did not restore normal transport rates of tNH 3 from within the cell in the lumen ( 12, 15 ). In the present study, microperfusion of S3 with a low-sodium buffer containing amiloride markedly inhibited luminal tNH 3 secretion rates. The NHE3 isoform is the predominant apical Na + /H + exchanger in the proximal tubule and has been localized in the apical membrane of the S1 and S2 segments ( 2, 3 ). Not all groups have been able to identify the NHE3 isoform in the S3 segment ( 2, 3 ). Thus transport differences between S2 and S3 proximal tubule segments could result from differences in the expression of NHE3 and/or by differences in other tNH 3 transporters expressed in the two segments. For example, if NHE3 is absent from the S3 segment, the newly described NHE8 isoform, which is expressed throughout the proximal tubule, especially in the outer medullary region, could theoretically contribute to tNH 3 secretion in the S3 segment ( 10 ). At this time, the inhibitor and transport characteristics of the NHE8 are not known, and its regulation by acid-base disturbances is unknown. Another alternative pathway for tNH 3 secretion in the S3 segment is the parallel apical diffusion of NH 3, with H + secretion mediated by an H + -ATPase. However, in our previous studies in the S2 proximal tubule segment ( 15 ) and in the present study in S3 segments, artificially reducing the pH of the luminal fluid by markedly lowering the perfusion pH did not completely restore tNH 3 secretion rates in the lumen when the segments were perfused with a low-sodium perfusion buffer containing amiloride.

In summary, S3 proximal tubule segments from acidotic mice display enhanced rates of tNH 3 production and secretion when ANG II is present in the luminal fluid. The enhancement of tNH 3 production and secretion rates by ANG II is inhibited by the AT 1 receptor blocker losartan. In S3 segments, the enhancement of luminal tNH 3 secretion rates by ANG II may be primarily driven by enhanced tNH 3 production rates that increase the transportable pool of tNH 3. These studies taken with studies demonstrating the presence of ANG II in the luminal fluid in vivo suggest a contributory role of the S3 segment in enhancing urinary tNH 3 excretion in response to acidosis.

GRANTS

This work was supported by Medical Research Funds from the Department of Veterans Affairs.

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作者单位：Nephrology Section, Medical and Research Services, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles 90073; and Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095