Acidification in the medullary collecting duct following ureteral obstruction

Kidney International, Vol. 29 (1986), pp. 1167—1/7/

Acidification in the medullary collecting duct following ureteral obstruction CARLA RIBEIR0 and WADI N. SuKI Renal Section. Department of Medicine, Baylor College of Medicine, The Veterans Administration Hospital and The Methodist Hospital, Houston, Texas, U.S.A.

Acidification in the medullary collecting duct following ureteral obstruc-

tion. A defect in urine acidification has been described in obstructive uropathy. Since the collecting tubule from the inner stripe of the outer medulla (OMCTi) is the major site for distal acidification, isolated OMCTi nephron segments from control rabbits and from rabbits after 24 hr of unilateral (UUO) or bilateral (BUO) ureteral obstruction were

studied. Tubules were perfused (4 nliter/min) and bathed with an artificial solution resembling rabbit serum ultrafiltrate, and 3H inulin was incorporated in the perfusate as a volume marker. Water absorp-

tion (Jv) was —0.03 0.03 nliter mm' min 'in control tubules, and 0.12 nliter was significantly (P < 0.05) negative in UUO (—0.48 mm -' min ')and BUO (—0.29 0.07 nliter/mm min ')tubules, as a result of an inulin leak. Bicarbonate absorption (JHco3) in control tubules was 11.61 1.21 pmole mm' min' and was significantly lower in UUO tubules (7.59 1.09 pmole mm' min, P < 0.05). JHCO3 in BUO tubules although tower than control (7.96 2.75 pmole • min') did not achieve statistical significance because of a high

tween urine and blood [6, 7]. Failure of this system results in the

development of distal renal tubular acidosis (DRTA), a syndrome which is well characterized clinically [6—10], and, among

its features, is an inappropriately high urine pH relative to the degree of acidosis [6—9].

Previous investigations have demonstrated an acidification defect following the release of ureteral obstruction [11—13]. The present isolated tubule microperfusion studies were performed in order to determine the effect of 24 hr unilateral and bilateral

ureteral obstruction (UUO and BUO respectively) on H + ion secretion by the rabbit OMCTi. Study of this model of impaired H ion secretion may help promote our understanding of the pathogenetic mechanism(s) involved in DRTA in humans.

degree of heterogeneity among tubules. To determine whether the acidification disorder was due to a gradient or capacity defect, the ability of the tubules to lower HCO3- concentration (HCO3) at low

Methods

Male New Zealand rabbits weighing 1.5 to 2.5 kg were used

rate of perfusion (1 nliter/min) was examined. No difference in HCO was found among the three groups being 8.98 0.54, 9.95 1.76, and 8.93 2.19 mmole in control, UUO and BUO tubules respectively. We conclude that ureteral obstruction results in an acidification defect due to a reduced capacity for H secretion.

in all experiments. They were fed Purina Rabbit Chow and allowed tap water ad lib, Complete unilateral and bilateral ureteral obstruction (UUO and BUO respectively) was performed, with sterile technic under general anesthesia with intravenous pentobarbital (Nembutal sodium, Abbott Laboratories, North Chicago, Illinois, USA) 25 mg/kg, by tying a 4-0

The distal nephron of the mammalian kidney plays an important role in regulating acid—base homeostasis [1-4]. Its most

cal junction. The animals were permitted to recover from

important function, the titration of luminal buffers and the

silk suture around the left or both ureters above the ureterovesi-

anesthesia and were returned to their cages, with free access to food and water. After 24 hr, they were sacrificed by decapita-

delivery of bicarbonate to the peritubular environment, results tion and the left kidney from either obstructed or control in the excretion of H ions and generation of bicarbonate to animals was removed and sliced for dissection and microperfurestore that which was lost in titrating metabolic acid loads [5]. sion, as previously described [14]. Briefly, 1 to 2 mm coronal Recently, Lombard, Kokko, and Jacobson demonstrated that slices were placed in a chilled artificial medium similar in substantial rates of bicarbonate reabsorption occur in the outer composition to plasma ultrafiltrate, with fetal calf serum (5%

medullary collecting tubule obtained from the inner stripe vol/vol) added to reduce adherence of the tubules to the (OMCTi) from normal rabbits, whereas there was no net dissecting dish, Segments were dissected free hand with the aid bicarbonate transport in the cortical collecting tubule (CCT) of a dissecting microscope and fine forceps. Isolated tubules from normal animals (4), which indicates that the OMCTi is a were transferred to a thermostatically controlled lucite chammajor nephron site of distal urinary acification. ber, connected to concentric glass pipettes, and perfused at The terminal nephron has a low capacity, high gradient 37°C. The tubules were perfused and bathed with an artificial system for secretion of less than 10% of the H ions secreted by

the kidneys, and can generate a maximal PCO2 gradient be-

solution resembling plasma ultrafiltrate with the following composition (in mM); NaCI 105; NaHCO3 25; KC1 5.0; Na2HPO4 0.5; Na acetate 10; Mg SO4 1.0; CaCl2 1.8; Glucose 8.3; alanine

Received for publication December 12, 1984 and in revised forms April 8, September 4 and November 7, 1985

5.0. Methoxy-3H-inulin (ICN Pharmaceuticals, Inc., Irvine, California, USA) was exhaustively dialyzed and added to the perfusate to serve as an impermeant volume marker. The bath

© 1986 by the International Society of Nephrology

was exchanged at a rate of 0.5 mliter/min and all solutions were

1167

Ribeiro and Suki

1168

bubbled with 5% C02195% 02 to achieve a pH of 7.4. After the initiation of perfusion, the tubules were allowed to stabilize for 60 mm, and then the following measurements were made: 1) Vo (collection rate), by timing the collection of fluid emerging from the tubule into constant volume pipettes; 2) Vi (perfusion rate), calculated from the 3H-inulin activity in perfused and collected

fluid; 3) Jv (volume flux), calculated from the difference between Vi and Vo; 4) HC03, calculated from the difference in total CO2 content between perfusate and collected fluid; 5) JHCO, calculated from the total CO2 content of perfused and collected fluid by the following equation: '}-1C03

—

0

L

as it has been previously demonstrated [41, and

confirmed in this study in a series of 10 control experiments where Jv was —0.03

P 0.05

r 0.05 60

0

C

50

— 40

E

—30 E E

—20

Vo (Ci - Co)

where Vo is the collection rate, Ci is the total CO2 content in the perfusate, Co is the total CO2 content in the fluid collections and L is the tubule length. This simplified equation presupposes that

Jv =

F

C —10

> -)

0• Fig. 1. Fluid absorption in ,nedullar collecting tubules from control (, N = 10), unilateral (UUO) (U. N = /5), and bilateral (BUO) (, N = 18) ureterml obstruction animals. Note that in both experimental groups there was an apparent negative iv.

0.03 nliter/min/mm.

Most tubules were perfused at a rate of 4 to 5 nliter/min, which is the physiological rate for this segment. In some important to establish that this parameter was maintained experiments perfusion rate was reduced to 1 nliter/min in order to investigate the ability of the tubular cells to generate a steep

H ion gradient. All fluid collections were deposited on a small siliconized petri dish, and covered completely with 3 to 4 mliter of mineral oil that had been previously equilibrated with water and bubbled with 6.14% CO2 and 93.86% 02. Aliquots of fluid were then withdrawn for determination of total CO2 content by microcalorimetry [15], and for liquid scintillation counting. In ten UUO and BUO experiments, timed collections of the bathing solution were also made and aliquots counted for their inulin content. In fourteen control and BUO experiments blood, obtained at the time of sacrifice, was analyzed for acid—base parameters. The data were expressed as the mean of 3 to 5 collections for each experiment in the control, UUO and BUO groups, and the group data given as mean SE. Statistical analysis was carried out using unpaired t-test and significance was accepted if the P value was < 0.05. Results Acid—base status

Inspite of 24 hr of bilateral ureteral obstruction, the blood pH 1.2 mmole/liter) did not 0.02) and total CO2 (26.2 (7.40 differ significantly from those of control rabbits (7.37 0.01

and 28.6 1.1 mmole/litcr, respectively). It is not likely, therefore, that unilateral obstruction could have induced a disturbance of acid—base balance.

Studies of bicarbonate absorption At the physiological perfusion rate of 4 to 5 nliter/min, the following parameters were measured in the three groups: Vo, Jv, AHCO3, and JHCO. The values for Vo in control, UUO and BUO tubules were, respectively, 4.48 0.14, 4.43 0. 10 and 4.50 0.13 nliter/min, with P values of 0.77 (NS) for UUO and 0.92 (NS) for BUO experiments. Since the rate of acidification

may be dependent upon the rate of delivery of buffer, it is

constant during all experiments.

Figure 1 depicts water flux in control, UUO and BUO tubules, and shows a mean Jv of —0.03 0.03 nliter/min/mm for control, —0.48 0.12 nliter/min/mm for UUO and —0.29 0.07 nliter/min/mm for BUO tubules (P < 0.05 for both UUO and BUO experiments). This small but significant negative Jv found in experimental tubules from UUO or BUO represents an

inulin leak in these tubules exposed to a high intraluminal pressure for 24 hr, some tubules being more affected than others. In the experiments on UUO and BUO tubules in which

bath collections were carried out, a mean of 10.48

2.52

percent of the perfused inulin was recovered in the bath. This value is consonant with the negative iv in the two groups being 6.6 and 10.7 percent of the perfusion rate, respectively. In fact, a positive correlation was observed between the percent inulin leak and the negative Jv(r = —0.781, P < 0.05). Figure 2 shows the HCO3 data from control, UUO and BUO

experiments. As can be observed in this diagram, control tubules were able to acidify the luminal fluid significantly more

than UUO tubules, with respective HCO3 values of 4.78 0.49 mri and 3.11 0.42 mt (P < 0.05), which would be expected from a distal RTA model of obstructive uropathy. On the other hand, the mean HCO3 from BUO tubules was 3.62

1.11 which, although lower than control, did not achieve statistical significance (P = 0.46, NS) because of a high degree of intertubule heterogeneity. JHCO, from control, UUO and BUO groups is shown in Figure 3. The respective JHCO1 for control and UUO experiments was 11.61 1.21 pmoles/mm/min and 7.59 1.09 pmoles/mm/min (P < 0.05), with UUO tubules showing an acidification defect in

comparison to control. With the buffer load delivered by a perfusion rate of 4 to 5 nliter/min, both a defect in the capacity to secrete protons and a defect in the ability to generate a steep H gradient can yield similar findings. JHCO, in BUO tubules was also reduced to 7.96 2.75 pmoles/mm/min. However, unlike UUO, this was not significantly different from control (P = 0.35, NS) owing to a high degree of heterogeneity among tubules in this group.

1169

Obstruction-induced acidification defect

P>0.05

NS

NS —,

I—P < 12

5 10

4 E

00 I -1

3

E

8

0 C)

6

I -1

2

4 2

C'

0

Fig. 2. The lowering of bicarbonate concentration in collected fluid from medullary collecting tubules of control, unilateral (UUO) and bilateral (BUO) ureteral obstruction animals. Note the greater HCO3 in control than in experimental tubules. (Refer to Fig. I for symbols).

P>0.05 <0.05-i 12 C

4

conditions of this study simulate the situation which would be found after the release of obstruction in in vivo preparations. As opposed to Hanley's observations [16] where, studying

2

different nephron segments after 24 hr UUO and BUO, he found no differences in intrinsic function after the release of

carried out to more directly investigate this problem. The

c0

a>

0 0 U

I

-)

In this isolated tubule microperfusion study of obstructive nephropathy, tubules dissected from kidneys that had been obstructed for 24 hr prior to study were investigated. Since

8

E

E

Discussion

studies evaluating the effect of an acute increase in intraluminal pressure on H + ion secretion by the OMCTi have to date been indirect, the present in vitro study of this nephron segment was

10 E

Fig. 4. The lowering of bicarbonate concentration in collected fluid from medullary collecting tubules perfused at slow rate of I nliter min'. Note that HCO3 is not different in control (N = 5), unilateral (UUO) (N = 5) and bilateral (BUO) (N = 7) ureteral obstruction animals. (Refer to Fig. I for symbols).

0

Fig. 3. Bicarbonate absorption by medullary collecting tubules from control, unilateral (UUO) and bilateral (BUO) ureteral obstruction animals. Note the greater JHCO> in control than in experimental animals.

(Refer to Fig. I for symbols).

BUO when compared to UUO, our data show a high degree of heterogeneity in the function of the OMCTi segment after 24 hr of bilateral ureteral obstruction. Heterogeneity of tubular function after acute unilateral or bilateral ureteral obstruction has been an important finding in a number of other studies [17—19].

Harris [17] demonstrated filtration in only 40% of surface nephrons and 12% of juxtamedullary nephrons after release of unilateral ureteral obstruction in rats. In another investigation,

Studies of H gradient

Buerkert et al [18], studying the effect of acute unilateral

perfusion rate, and suggests a defect in the capacity for H

obstruction on deep nephron function in rats, observed filtration in 41% of surface nephrons and 33% of deep nephrons. Investigating the functional defect after BUO in rats, Jaenike [19] showed that about one-fourth of the superficial nephrons were severely affected, and inulin was found to leak from the lumen into the peritubular capillaries which confirms, again, the presence of heterogeneity after elevated intraluminal pressure. In the present study, we found that tubules from UUO and, especially, from BUO experiments exhibited a significant degree of variability with respect to water flux and the ability to acidify the luminal fluid. In seven of 15 UUO tubules Jv was outside the range of normal values. With respect to HCO3, whereas nine of ten control tubules had values greater than 3

secretion to account for the defect in JHCO, observed earlier.

mmoles, eight of 15 UUO tubules had values that fell below this

These

studies were carried out at a perfusion rate of 1

nliter/min in order to determine the ability of the tubular cells to

generate a steep H + gradient between the lumen and bath (or cytosol) at this very low perfusion rate. Figure 4 shows the HCO3 (mM) at I nliter/min perfusion rate in control (8.98 1.76) and BUO (8.93 0.54), UUO (9.95 2.19) experiments (in these experiments only HCO3 was measured). As can be seen, there was no significant difference between control and UUO or control and BUO experiments (the respective P values were 0.59 and 0.99), which indicates an intact ability of the tubular cells to generate a steep H gradient at this very low

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Ribeiro and Suki

level. Similar variability was observed in the BUO group with an inability to generate a steep H + ion gradient across the distal eight of 18 tubules falling outside the range of control tubules in nephron epithelium. Another possibility was that a steep H either parameter examined. The high degree of tubular hetero- ion gradient could not be maintained because of increased geneity observed can be explained by the increased intraluminal backleak of secreted H + ions due to an altered distal nephron pressure and the change in medullary plasma flow. Solez and membrane permeability [7], which would also cause a gradient colleagues [20] found a gradual decrease in inner medullary defect. It is now clear that a reduced capacity to secrete H + will plasma flow after 18 hr UUO or BUO, greater in BUO than also result in distal RTA [6], and recently, the studies of Arruda UUO, as well as a higher ureteral pressure in BUO experi- and colleagues in acidifying membranes have demonstrated a ments. In the same study [201, tubular necrosis was demon- voltage—dependent defect in distal acidification caused by lithstrated in portions of the inner and outer medulla after 18 hr of ium [28] or amiloride [29]. An important aspect of our investiUUO or BUO and poor filling of vasa recta in these regions of gation was the fact that when the perfusion rate was lowered the kidney. Harris and Yarger [171, studying the effect of UUO from 4 to 5 nliter/min to 1 nllter/min, we could demonstrate that in rats, concluded that the peritubular plasma flow in the outer the intrinsic ability of the tubular cells to increase the H + ion cortex was better preserved than in the juxtamedullary cortex gradient between lumen and bath (or cytosol) was intact in both or outer medulla. Another interesting aspect that should be UUO and BUO tubules. From this observation, one can conpointed out is that the pressure in the peritubular capillaries was clude that the impaired distal acidification seen in this model of also elevated when intratubular pressure was increased [21]. obstructive nephropathy is caused by a defect in the capacity of Our finding that BUO tubules were not as uniformly affected as the tubular cells to secrete H At high perfusion rate the UUO tubules, with respect to their ability to secrete H ions, delivery of buffer to the tubular cells must have exceeded their was similar to that of Walls and colleagues [11] who, after the capacity to secrete H leading to the decreased HC03 obunilateral release of bilateral ureteral obstruction in rats, dem- served in that group of studies. At the slow perfusion rate the onstrated a less marked acidifying defect in this group than that HCO3 generated was approximately 36% of the concentration of HCO3 in the bath. This is over threefold the measured seen after relief of UUO. The integrity of the collecting tubules with respect to its proportion of inulin leak and, thus, it was possible for these

electrolyte and water transport properties after relief of tubules to generate a gradient for HC03 in spite of the obstruction is not very clear. Micropuncture studies of the leakiness of the epithelium. It may be argued that a channel papillary collecting duct show normal reabsorption of water and permeable to a macromolecule, such as inulin, may be more salt [22, 23]. On the other hand, Sonnenberg's data [24j using microcatheters in the medullary collecting tubule show that, following the release of obstruction, this nephron segment no longer reabsorbs water, and salt and water secretion may be

observed. In our studies there was a significant difference

permeable to a smaller molecule such as bicarbonate. However,

bicarbonate is a charged species and, if the channel were similarly charged, impedence of its permeation can result. It is also possible that, by lowering the perfusion rate to I nliter/min and decreasing the tubular diameter, channel diameter becomes

also reduced thereby abolishing a backleak of HC03 which may have existed at the higher perfusion rate. Finally, it is experiments, there was an apparent negative water flux which possible that because of tubule heterogeneity a defect was not was most likely due to an increase in the permeability of the encountered in the small number of tubules examined. Whattubular epithelium to inulin, A leak of inulin into the bathing ever the reason(s) for the discrepancy between a significant solution of equal magnitude to the negative water flux was bicarbonate gradient and a leak of inulin, the tubules that were between control, on the one hand, and UUO and BUO exper-

iments, on the other, regarding Jv. In the UUO and BUO

demonstrated. This is consistent with the findings of McDougal examined were able to generate a normal gradient for bicarbonand Wright [25] where rats subjected to ureteral obstruction of ate at low bicarbonate loads while tubules presented with four30 hr duration had increased permeability to mannitol and inulin to fivefold the bicarbonate load generated a significantly lower in the collecting duct. Similarly, following renal venous and bicarbonate gradient. This constellation of findings is compatiureteral constriction with increased intrarenal and intratubular ble with a limitation in the capacity of these tubules to reabsorb pressure, Lorentz, Lassiter and Gottschalk [26] demonstrated a bicarbonate caused by obstruction.

mannitol recovery significantly less than 100% after

In conclusion, using the model of obstructive nephropathy, microinjections into distal convolutions, suggesting a defect we have demonstrated that, after 24 hr of UUO or BUO, there beyond this nephron site. After elevation of intratubular pres- is a high degree of tubular heterogeneity and tubular damage. sure, Lorentz, Lassiter, and Gottschalk [26] demonstrated an Furthermore, we have demonstrated the existence at the tubuabnormally increased permeability to volume markers inversely

related to molecular size, which suggests diffusion through

lar level of an acidification defect following 24 hr of U UO which appears to be due to a defect in the capacity of the OMCTi cells

aqueous channels of increased diameter and postulated that the to secrete H tight junction and lateral intercellular spaces would be involved Acknowledgments in the pathway. In our model of elevated intratubular pressure it is possible that a similar increase in channel diameter ocThe data were presented at the Annual Meeting of the American curred after 24 hr of UUO or BUO, which in turn may have led Society of Nephrology, Washington, D.C., December, 1984. Work

to the leak of inulin to explain the apparent negative Jv in

supported by Grant No. AM-2 1394 from the National Institutes of

Health, U.S. Public Health Service, and in part by funds from the experimental tubules. Veterans Administration. Until a few years ago, all cases of distal RTA were thought to result from a similar mechanism. The then prevailing hypotheReprint requests to Dr. Wadi N. Suki, Renal Section, The Methodist sis proposed by Seldin and Wilson [27] attributed distal RTA to Hospital, 6565 Fannin, Houston, Texas 77030 USA.

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Obstruction-induced acidification defect

and study of fragments of single rabbit nephrons. Am J Physiol

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