INTRODUCTION

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THE AVIAN KIDNEY HAS a highly complex organization involving two major nephron populations, with gradations between the two (3, 9, 26). The most superficial cortical nephrons (often referred to as reptilian-type nephrons) (13) are small, lack loops of Henle, and empty at right angles into collecting ducts (6, 26, 27). The deepest medullary nephrons (often referred to as mammalian-type nephrons) (13) are large, with complex convoluted proximal tubules and long loops of Henle lying parallel to collecting ducts (6, 26). The gradation between the reptilian-type and the mammalian-type nephrons is made up of transitional nephrons. These have relatively straight proximal tubules and short-looped intermediate segments that do not lie parallel to collecting ducts (6, 26).

The role of the proximal tubules of these different types of nephrons in the maintenance of acid-base homeostasis for the animal as well as the factors involved in the maintenance of pH within the tubule cells themselves [intracellular pH (pH_i)] are only beginning to be explored. This is despite the fact that the proximal tubules of birds, like those of other tetrapod vertebrates, must deal with systemic dietary acid or base loads while maintaining their own intracellular acid-base homeostasis. Some in vivo micropuncture studies on superficial loopless reptilian-type avian nephrons (19, 20) and in vitro microperfusion studies on short-looped transitional avian nephrons (7) indicate that along the proximal tubule there is little acidification of luminal fluid, that net bicarbonate reabsorption proceeds at the same rate as net fluid reabsorption, and that net fluid reabsorption is not dependent on bicarbonate reabsorption.

An initial study on proximal tubules from chicken short-looped transitional nephrons indicated that resting pH_i is higher than that generally found under similar circumstances in rabbit and snake proximal tubules and that maintenance of pH_i is dependent on both Na⁺- and Cl-coupled acid-base fluxes at the basolateral membrane (14). pH_i studies were undertaken initially on proximal tubules from short-looped transitional nephrons because they were the only group of avian proximal tubules that we were able to tease from fresh tissue. Recently, we have learned to tease proximal tubules from superficial loopless reptilian-type avian nephrons. Because these superficial loopless reptilian-type nephrons in the avian kidney are so different structurally from the deeper short-looped transitional nephrons, we undertook to examine pH_i and its regulation in proximal tubules from this population. The results indicate that in proximal tubules from these nephrons 1) resting pH_i is lower than resting pH_i in proximal tubules from short-looped transitional nephrons and is about the same as resting pH_i in snake and rabbit proximal tubules and 2) basolateral mechanisms for acid-base fluxes involved in pH_i regulation are different from those in proximal tubules from short-looped transitional nephrons.

	METHODS

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Preparation of isolated renal tubules. Male and female White Leghorn chickens, 1-3 mo old, were decapitated. Their kidneys were flushed in situ via the aorta with chilled (4°C) avian Ringer solution, quickly removed, and placed in the same solution on ice (see below for composition of solutions). Tubules were dissected from thin, vertical slices of kidney without the aid of enzymatic agents, as described previously (7). We normally used only one tubule segment from each bird (total of 51) in these experiments. The dissections were performed in chilled, oxygenated medium in a dissection dish maintained on ice. As noted above, avian nephrons vary from simple, superficial cortical nephrons without loops of Henle to deep medullary nephrons with long loops of Henle. Proximal segments (~500 µm in length) were dissected from superficial loopless reptilian-type nephrons. In our previous work (7, 14), we used only proximal tubules from short-looped transitional nephrons because they were the only proximal segments that we were able to tease from avian tissue without the aid of enzymatic agents. However, as pointed out above, we have now learned to tease out proximal tubules from loopless reptilian-type nephrons to use in the nonperfused state for measurements of pH_i. The lumina of proximal tubules from these nephrons, like the lumina of proximal tubules from short-looped transitional nephrons (14), collapse rapidly so that no fluid is detectable in them. Dissections were performed in chilled medium (4°C), but all experiments were performed at 37°C.

Ringer composition. The components of the avian Ringer solutions used in these studies are shown in Table 1. All solutions were buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Solution 1 is the basic solution established previously for flushing the kidneys and for dissection of avian renal tubules (7). As noted in our previous work (7, 14), although the osmolality of this solution containing sucrose (Table 1) is substantially above the normal plasma osmolality (24), we found it to be the best solution for dissecting these tubules in vitro. There was no apparent change in cell volume of tubules maintained in this solution. Solution 2, which was identical to solution 1 except for the removal of the sucrose (Table 1), was used for the incubation period with the pH-sensitive fluorescent dye (see below). The sucrose was removed to avoid possible interference with the dye uptake or calibration. Solution 3 was the standard solution used for the control pH_i measurements and for studies with the addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and ethylisopropylamiloride (EIPA). This solution was identical to solution 2 except that all extra organic substrates (except glucose) were replaced with NaCl to prevent any possible effect of these substrates on pH_i. Solutions 4-8 involved modifications in solution 3 designed to examine the effects of Na⁺, Cl, K⁺, and Ba²⁺ on pH_i (see RESULTS). The pH of each solution was adjusted to 7.4 at room temperature with 1 N NaOH, 1 N KOH, or tris(hydroxymethyl)aminomethane base, as appropriate. When 20 mM NH₄Cl was present in the medium, the concentration of NaCl was reduced by an equimolar amount to maintain the osmolality and ionic strength approximately constant. The osmolality of all solutions except solution 1 was ~290 mosmol/kgH₂O (Table 1) and was checked regularly with a vapor pressure osmometer. The solutions were continuously bubbled with 100% O₂ and were assumed to be nominally free in the absence of tissue.

Measurement of pH_i in single renal tubules. We used the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to measure pH_i in a manner similar to that described by others and used previously by us (14, 15, 17). For these measurements, we used a dual-wavelength spectrofluorimeter built around an Olympus IMT-2 inverted epifluorescence microscope. A 100-W mercury arc lamp was used as an excitation source, and specific excitation wavelengths were selected by a filter wheel mounted to the shaft of a high-speed motor. Wavelength-specific filters for measurement of pH using BCECF were centered at 445 (isobestic wavelength) and 495 nm. The selected excitation light was directed to the sample by a matched dichroic mirror. To prevent photo damage to the dye-loaded cells from the excitation light, two neutral density filters (ND 2, Oriel) were placed in front of the illumination site. The emitted fluorescent light passed through the dichroic mirror and a wavelength-specific emission filter (530 nm for BCECF). The fluorescent light emitted from a selected region of the sample was collected by a Hamamatsu HC120-03 photomultiplier tube operated in photon-counting mode. The dark current of the photomultiplier tube was zeroed out with a discriminator. Collection of fluorescent light was synchronized to the wheel rotation and excitation filter position. Synchronization, speed selection, and data collection were controlled by a microcomputer running custom software. The integrated average of 30 measurements/s was collected at 1-s intervals.

Individual tubules were held in an appropriate bathing chamber on the stage of the microscope and incubated in solution 2 containing the acetoxymethyl ester (AM) form of BCECF (6 mM) (first dissolved in dimethyl sulfoxide) for ~45 min at 25°C. The AM form of BCECF readily enters the cells where the ester is cleaved by nonspecific esterases yielding the impermeant, fluorescent form of the dye. After the loading period, the bath was replaced with dye-free solution 3. The tubule and bathing chamber were rinsed several times with this dye-free solution to remove remaining extracellular dye before an experiment was begun. For collection of fluorescent light, the microscope was equipped with a Zeiss ×63 Neofluor oil-immersion objective (1.25 numerical aperture). Wavelength-specific fluorescence was collected as described above over a 22- to 32-mm-diameter area of nonperfused tubule. The ratio of fluorescence at 495/445 nm was then used as a measurement of pH_i to eliminate the influences of changes in dye content or in cell shape or volume. Calibration of the pH sensitivity of intracellular BCECF was performed for each tubule at the end of each experiment. This involved monitoring the 495/445 nm ratio at various values of pH_i by incubating the tubule in a solution with high K⁺ containing the ionophore nigericin (13 mM) (which exchanges K⁺ for H⁺ and sets pH_i to approximate extracellular pH) (25). The calibration curve was linear between pH 6.5 and 8.0. Autofluorescence was insignificant compared with the fluorescence from BCECF and was taken into account by the calibration procedure.

Chemicals. Nigericin and DIDS were purchased from Sigma. BCECF and EIPA were purchased from Molecular Probes. All other chemicals were purchased from standard sources and were of the highest purity available.

Statistics. Values are summarized as means ± SE. Significant differences between values were determined with Student's t-test for paired or unpaired data, as appropriate. Linear regression analyses were performed for calibrations as required. In all analyses, differences were considered statistically significant when P < 0.05.

	RESULTS

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Control measurements of pH_i. We measured the control resting pH_i in isolated proximal tubules from chicken loopless reptilian-type nephrons before studying changes in pH_i in response to various treatments. As summarized in Table 2, the resting pH_i was ~7.2 or below.

Response of pH_i to exposure to NH₄Cl pulse. To alter pH_i, we exposed tubules for 30-60 s to 20 mM NH₄Cl in the bathing medium (14, 15, 22). NH₃ diffuses across cell membranes much more readily than NH⁺₄ (10). Therefore, NH₃ should rapidly enter the tubule cells and combine with free intracellular H⁺ to form NH⁺₄ and alkalinize the cell interior. In principle, pH_i should increase until the intracellular NH₃ concentration ([NH₃]_i) is equal to the extracellular NH₃ concentration. NH⁺₄ should enter the cells more slowly than NH₃, leading to a gradual decrease in pH_i over the exposure period. When NH₄Cl is then removed from the bathing medium, free NH₃ should diffuse rapidly from the cells, leaving behind free H⁺ and producing rapid acidification of the cell interior. The results of this process are summarized for all individual tubules studied in Table 2. The results are also shown for individual proximal tubules from chicken loopless reptilian-type nephrons in Figs. 1-4. The expected pattern was observed, with both the maximum pH_i in the presence of NH₄Cl (~7.7) and the minimum pH_i after removal of NH₄Cl (~6.9) being significantly different from the resting pH_i (Table 2).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Intracellular pH (pHi) in single proximal tubule from chicken superficial loopless reptilian-type nephron during various modifications in bathing medium. Boxes above tracing indicate when medium is control Ringer (Control) or Ringer with addition of NH4Cl.

Rate of pH_i change, intrinsic buffering capacity, NH₃ flux across the basolateral membrane, and permeability of basolateral membrane to NH₃ during NH₄Cl pulse experiments. To examine more quantitatively the factors involved in the changes in pH_i during the NH₄Cl pulse experiments, we measured the rate of change of pH_i (dpH_i/dt) and calculated the intrinsic buffering capacity (_i), NH₃ flux across the basolateral membrane (J_NH3), and the permeability of the basolateral membrane to NH₃ (P_NH3) during the initial alkalinization (addition of NH₄Cl to the bath) and acidification (removal of NH₄Cl from the bath). These calculations were performed as in our previous studies (14, 15) and are described briefly below.

(1)

2

1

(2)

(3)

View this table: [in this window] [in a new window]   Table 3.   dpHi/dt, i, JNH3, and PNH3

Effects of Na⁺, Cl, or Na⁺ and Cl removal on rate of recovery from acid pH_i to control resting pH_i. We examined rate of recovery of pH_i (dpH_i/dt) from the acid value after removal of NH₄Cl to the control resting value in these renal proximal tubules from chicken loopless reptilian-type nephrons. The average control value for this recovery rate is given in Table 4, in which it is compared with the values for proximal tubules from chicken short-looped transitional nephrons and snake and rabbit nephrons obtained in our previous studies (14, 15) (see DISCUSSION). The control recovery is also shown for individual tubules in Figs. 1-4.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   pHi in single proximal tubule from chicken superficial loopless reptilian-type nephron during various modifications in bathing medium. Designations in boxes above tracing indicate same changes as in Fig. 1 with the addition of Na+-free Ringer (0 mM Na+), Cl-free Ringer (0 mM Cl), and Na+- and Cl-free Ringer (0 mM Na+, Cl).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   pHi in single proximal tubule from chicken superficial loopless reptilian-type nephron during various modifications in bathing medium. Designations in boxes above tracing indicate same changes as in Fig. 1 with the addition of 1 mM ethylisopropylamiloride (EIPA) and 0.25 mM DIDS.

Effects of high K⁺ concentration or presence of Ba²⁺ on the rate of recovery from acid pH_i to control resting pH_i. As noted above, at least one basolateral mechanism that might be involved in regulation of pH_i is the electrogenic Na⁺-- cotransporter that moves out of the cells. Its continued function would tend to slow the rate of pH_i recovery. Because it is electrogenic, its function and thus its ability to delay the rate of recovery would be reduced if the basolateral membrane potential were reduced. To examine this possibility, we undertook maneuvers to reduce the basolateral membrane potential. Although we did not measure membrane potential directly in these studies, we assumed that a high concentration of K⁺ in the bathing medium or the addition of Ba²⁺ to the bathing medium would reduce the membrane potential, as they do in renal tubules from other species (15). Therefore, we examined the effects of increasing the K⁺ concentration in the bathing medium to 75 mM (solution 7, Table 1) and of adding 5 mM Ba²⁺ (solution 8, Table 1) to the bathing medium on the rate of recovery of pH_i. The results are shown in Table 5 and Fig. 4. As would be expected if the mechanism suggested above were slowing pH_i recovery, increasing the K⁺ concentration to 75 mM produced a significant increase in the rate of recovery (Table 5, Fig. 4). However, in opposition to this expectation, the addition of 5 mM Ba²⁺ to the bathing medium had no effect on the rate of recovery (Table 5, Fig. 4). Because of the lack of effect of 5 mM Ba²⁺, in a few experiments we also examined the effect of the addition of 10 mM Ba²⁺ to the bathing medium and the effect of the combination of 10 mM Ba²⁺ and 75 mM K⁺ in the bathing medium on the rate of recovery of pH_i. The addition of 10 mM Ba²⁺ had no effect, and the combination of 10 mM Ba²⁺ and 75 mM K⁺ produced the same increase in the rate of recovery as 75 mM K⁺ alone (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   pHi in single proximal tubule from chicken superficial loopless reptilian-type nephron during various modifications in bathing medium. Designations in boxes above tracing indicate same changes as in Fig. 1 with the addition of 5 mM Ba2+ and high K+ (75 mM).

Effects of removal of Na⁺, Cl, or both Na⁺ and Cl, of high K⁺ concentration, and of the addition of EIPA, DIDS, and Ba²⁺ on resting pH_i. In view of the effects of some of these treatments on the rate of recovery of pH_i, we also examined the effects of all of them on the resting pH_i. The results are summarized in Table 6. Removal of Na⁺ or of both Na⁺ and Cl produced an equal decrease in resting pH_i, whereas removal of Cl alone had no effect. Increasing the K⁺ concentration to 75 mM, which should have markedly reduced the basolateral membrane potential, produced a significant increase in resting pH_i. However, the addition of 5 mM Ba²⁺ to the bathing medium, which should also have reduced the basolateral membrane potential, had no effect on resting pH_i. DIDS had no effect on resting pH_i, but the addition of EIPA produced a significant increase in pH_i.

	DISCUSSION

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pH_i and its regulation in proximal tubules from chicken superficial loopless reptilian-type nephrons differ distinctly from those in proximal tubules from deeper short-looped transitional nephrons. In the present study, the resting pH_i (~7.20 or less) in proximal tubules from loopless nephrons in HEPES-buffered medium was significantly lower than the resting pH_i (~7.30-7.40) in proximal tubules from short-looped transitional nephrons in HEPES-buffered medium measured in our previous study (14). However, the resting pH_i in proximal tubules from these loopless reptilian-type nephrons was essentially the same as the resting pH_i in snake and rabbit proximal tubules in HEPES-buffered media, measured by others and by us with the same pH-sensitive fluorescent dye (BCECF) technique (11, 15, 16).

The general pattern of the response of pH_i to an NH₄Cl pulse in proximal tubules from these chicken loopless reptilian-type nephrons was similar to the pattern in proximal tubules from chicken short-looped transitional nephrons (14), snake nephrons, and rabbit nephrons (15). However, there were some quantitative differences in the information derived from these responses between the tubules in the present study and the chicken, snake, and rabbit tubules in the other studies (Table 3). In the case of proximal tubules from the two types of chicken nephrons, J_NH3 and P_NH3 were significantly lower in the loopless reptilian-type nephrons than in the short-looped transitional nephrons during both the initial increase in pH_i after the addition of NH₄Cl and during the decrease in pH_i after the removal of NH₄Cl (Table 3). Proximal tubules from chicken loopless reptilian-type nephrons, like proximal tubules from chicken short-looped transitional nephrons (14), tended to have a greater dpH_i/dt than snake proximal tubules and a lower _i than rabbit proximal tubules (15) (Table 3).

Of particular significance with regard to regulation of pH_i are the observations on the factors influencing the rate of recovery of pH_i from an acid value to the control resting, slightly alkaline value. The control rate of recovery is about the same in proximal tubules from these loopless reptilian-type nephrons as in proximal tubules from short-looped transitional nephrons (Table 4) (14). However, there are marked differences between proximal tubules from these two avian nephron populations in terms of factors influencing this rate of recovery. These differences probably relate to differences in mechanisms for acid and/or base fluxes across the basolateral membrane that may be involved in the recovery process (Fig. 5), although the exact mechanisms involved are not clear for either population of nephrons.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Diagram of suggested basolateral acid or base transporters (1-5) for regulating pHi in proximal tubules. Shaded transporter in gray indicates suggested new transporter.

In proximal tubules from the loopless reptilian-type nephrons, the observations that removal of Na⁺ from the bath depressed the rate of recovery but that EIPA addition did not affect it indicate that recovery probably does not involve Na⁺/H⁺ exchange at the basolateral membrane (Fig. 5; transporter 1). This is in striking contrast to proximal tubules from short-looped transitional nephrons in which both Na⁺ removal and amiloride addition depressed recovery to a similar extent, strongly suggesting that basolateral Na⁺/H⁺ exchange was an important factor in recovery of pH_i (14). On the other hand, the pattern observed in these avian reptilian-type nephrons is identical to that observed in true reptilian nephrons from snake kidneys, although the absolute rate of recovery in snake tubules, under these same conditions, is substantially lower than that in avian or mammalian tubules (Table 4) (15). Therefore, an amiloride-inhibitable basolateral Na⁺/H⁺ exchanger of the type demonstrated in proximal tubules from mammalian, amphibian, and avian short-looped transitional nephrons (4, 11, 14) does not appear to function in proximal tubules from reptilian or reptilian-type avian nephrons (15).

It is, of course, possible that a Na⁺/H⁺ exchanger completely insensitive to amiloride, such as that described in hippocampal neurons (21), exists in the basolateral membrane of reptilian or reptilian-type avian nephrons. However, although variations in sensitivity of the renal isoforms of Na⁺/H⁺ exchangers to amiloride and its analogs have been observed (8), no renal isoform completely insensitive to 1 mM EIPA has yet been reported. We have no explanation for the odd increase in resting pH_i observed with EIPA.

The absence of a basolateral Na⁺/H⁺ exchanger extruding H⁺ from the cells in reptilian and loopless reptilian-type avian nephrons might explain the lower resting pH_i in these two nephron types compared with that in short-looped transitional avian nephrons (14, 15). However, it does not explain why the resting pH_i in loopless reptilian-type avian nephrons and reptilian nephrons is the same as that in mammalian nephrons that do have a basolateral Na⁺/H⁺ exchanger (11, 15).

Another basolateral mechanism for recovery of pH_i that might be suggested is a DIDS-inhibitable Na⁺-coupled Cl/ exchanger moving into the cells, as described for mammalian tubules (Fig. 5; transporter 2) (2, 3, 11, 12). Some (largely produced by the tubule cells) must be present even in nominally -free media, such as those used in the present experiments, and Na⁺-dependent transporters can apparently function under these circumstances (11, 16). However, although the inhibition of recovery by Na⁺ removal or DIDS addition supports a mechanism of this type, the lack of effect of Cl removal does not. Therefore, this does not appear to be a functioning mechanism in chicken loopless reptilian-type nephrons, although it does appear likely in chicken short-looped transitional nephrons (14).

A basolateral DIDS-sensitive, electrogenic Na⁺-- cotransporter that moves out of the tubule cells, similar to that reported for amphibians and mammals (1, 5, 11, 18, 28) (Fig. 5; transporter 3) could also influence the pattern of pH_i recovery. Such a transporter is important in transepithelial reabsorption in these other species. However, it is not certain that this is the case in avian proximal tubules (7, 9). Moreover, the present study provides only conflicting evidence for such a transporter in these chicken loopless reptilian-type nephrons. Under control conditions in media nominally free of , movement out of the cells across the basolateral membrane by this mechanism should be greater than in media containing . Removing Na⁺ from the bathing medium should further enhance this efflux of , thereby reducing resting pH_i and the rate of recovery of pH_i. That is exactly what happened. Moreover, because this is an electrogenic process, depolarizing the basolateral membrane should increase resting pH_i and the rate of recovery of pH_i. This also is exactly what happened when the K⁺ concentration in the bathing medium was increased. However, the addition of Ba²⁺, which should have produced a similar effect on membrane potential, had no effect on resting pH_i or recovery of pH_i. This does not completely rule out such a transporter because there might be no Ba²⁺-sensitive K⁺ channels in the basolateral membrane of these tubule segments and Ba²⁺ might have had other effects (see below). However, the effects of DIDS in the present study also do not provide evidence for this transporter. In other species, this transporter is inhibitable by DIDS (5, 11), and such inhibition, by reducing efflux, should increase the rate of recovery of pH_i after acidification. Instead, in the current study, DIDS reduced the rate of recovery of pH_i. Thus there is no consistent evidence to support function of this transporter in proximal tubules from avian loopless reptilian-type nephrons.

There is no evidence for a basolateral DIDS-inhibitable, Na⁺-independent, electroneutral Cl/ exchanger like that described in other species (Fig. 5; transporter 4) (2, 3, 22) in proximal tubules from chicken loopless reptilian-type nephrons. If this were functioning, Cl removal should have decreased basolateral efflux from the cells (or actually produced basolateral uptake by reversing the transporter), thereby leading to an increased resting pH_i and an increased rate of recovery of pH_i. This did not happen. Again, these data contrast with our previous data on proximal tubules from chicken short-looped transitional nephrons (14).

A possible basolateral transport mechanism compatible with the data on rate of recovery, although not one described in other species, would be an electrogenic, DIDS-inhibitable Na⁺-coupled cotransporter that moves two or more ions into the cells (Fig. 5; transporter 5). The effects of Na⁺ removal, high K⁺ concentration, and DIDS addition on rate of recovery are all compatible with such a process. However, the lack of effect of DIDS on resting pH_i and the lack of effect of Ba²⁺ on either rate of recovery of pH_i or resting pH_i do not appear to support this proposed mechanism. Moreover, clear dependence of regulation of pH_i on the presence of in the absence of other buffers has yet to be demonstrated.

The lack of effect of Ba²⁺, however, as noted above, could simply indicate that there are no Ba²⁺-sensitive K⁺ channels in this membrane and that Ba²⁺ had no effect on membrane potential. This appears most likely in view of the observation that a high K⁺ concentration enhanced recovery of pH_i to the same extent whether Ba²⁺ was present or not. Because of the very small size and especially the extreme fragility of avian nephrons, we have not attempted to measure directly the membrane potential with microelectrodes as we have in other species (15). It is also possible that Ba²⁺ had an effect on acid or base flux other than that produced by its effect on membrane potential. For example, during the recovery of pH_i from acidification, acid might leave the cells as NH⁺₄, probably through K⁺ channels. If Ba²⁺ blocked K⁺ channels, it might not only reduce the membrane potential but also reduce or eliminate this NH⁺₄, flux and the two effects might cancel each other out. However, as noted above, the lack of effect of Ba²⁺ on the effect of high K⁺ suggests that K⁺ channels are not sensitive to Ba²⁺, and this alternative explanation involving NH⁺₄ flux for some of our observations appears unlikely.

In summary, there appear to be distinct differences between proximal tubules from chicken loopless reptilian-type nephrons and proximal tubules from chicken short-looped transitional nephrons in terms of basolateral mechanisms for the regulation of pH_i during maintenance in HEPES-buffered media. In proximal tubules from chicken short-looped transitional nephrons, a basolateral Na⁺/H⁺ exchanger clearly appears to be present and important (14). A Na⁺-dependent Cl/ exchanger, a Na⁺-- cotransporter, and a Na⁺-independent Cl/ exchanger (Fig. 5; transporters 2-4) also may play roles in the regulation of pH_i under these circumstances (14). Among these transporters, the Na⁺-dependent Cl/ exchanger appears most likely to be the major one (14). None of these mechanisms is clearly supported by the present data on proximal tubules from the chicken loopless reptilian-type nephrons, and an additional mechanism appears most likely. The reason for the apparent differences in basolateral regulation of pH_i between proximal tubules of these two nephron types is not apparent, although it may relate to differences in acid secretion and bicarbonate reabsorption as part of overall acid-base regulation. Although the information on proximal tubules from snake kidneys (15) is less complete than that on proximal tubules from chicken kidneys, the reptilian-type avian nephrons (with the exception of the effects of DIDS) appear to resemble true reptilian nephrons more than they do short-looped transitional avian nephrons.