Ammonia addition into the medullary collecting duct of the rat

Kidney International, Vol. 19 (1981), pp. 281—287

Ammonia addition into the medullary collecting duct of the rat HAROLD SONNENBERG, SURINDER CHEEMA-DHADLI, MARC B. GOLDSTEIN, BOBBY J. STINEBAUGH, ])ouGLAs R. WILSON, and MITCHELL L. HALPERIN Departments of Medicine and Physiology, University of Toronto, Toronto, Canada, and Department of Medicine, Baylor College of Medicine, Houston, Texas

The purpose of these studies was to examine

Ammonia addition into the medullary collecting duct of the rat.

The purpose of these studies was to determine if ammonia is

directly the handling of ammonium in the medullary

added directly to the medullary collecting duct of the rat, and if so, to estimate its quantitative contribution to ammonium excretion. Samples of fluid were obtained along the length of the mcd-

collecting duct and to estimate the amount of ammonia that may be added directly into this nephron segment. Although a previous investigation of this problem with the microcatheterization technique in the hamster [2] did show ammonia addition to the collecting duct, the quantitative aspects were not evaluated. Furthermore, micropuncture studies in the rat have produced conflicting results [3, 4]. Results indicate that about 40% of ammonium excreted in the urine is added directly to the medullary collecting duct of rats receiving a low or high pro tein diet.

ullary collecting duct by retrograde microcatheterization. To

document net addition of ammonium, we measured the ammonium concentration in tubular fluid by an enzymatic isotope technique, and we calculated the degree of fluid reabsorption from the ratio of the inulin concentration in tubular fluid and plasma. The ammonium concentration corrected for water reabsorption ([NH4]/[TF/P],) rose progressively from the beginning to the end of the medullary collecting duct, indicating net ammonia addition to Lhis nephron segment. By calculation, it appears that about 40% of the excreted ammonium reached the urine by direct addition of ammonia to the duct fluid. Addition d'ammoniaque dans le canal collecteur médullaire du

rat. Le but de cc travail a été de détecter une addition directe d'ammoniaque dans le canal médullaire collecteur du rat et, dans

cc cas, d'estimer sa contribution quantitative a l'excrétion

Methods

d'ammoniaque. Des échantillons de liquide ont été prélevés le long du canal collecteur médullaire par micro-cathétérisation. Atm d'évaluer une addition nette d'ammonium, la concentration d'ammoniaque dans le liquide tubulaire a été mesurée par une technique radio-enzymatique et la reabsorption de fluide a été évaluée au moyen du rapport de concentration de l'inuline (tubule/plasma). La concentration d'ammonium corrigCe pour la

Male Sprague-Dawley rats, each weighing between 240 and 320 g, were placed on low or high protein diets (Nutritional Biochemicals Co.) for 2 weeks prior to the experiments. Animals were pairfed by allowing the low-protein partner free access to food and limiting the high-protein partner to the same daily intake by weight. We anticipated that differences in ammonium excretion might be demonstrated in rats consuming diets with different protein loads. For renal clearance and collecting duct microcatheterization, the animals were anesthetized with

reabsorption d' eau ([NH4J/[TF/P},) augmente progressivement du debut a la fin du canal collecteur médullaire cc qui indique une addition nette d'ammonium dans cc segment du néphron. Le calcul montre qu'environ 40% de l'ammonium excrCtC provient d'addition directe au canal collecteur.

Ammonium excretion in the urine is the major means by which most species dispose of their daily hydrogen ion load. Ammonia is produced primarily in the renal cortex and is thought to enter the tubu-

mactin (100 mg/kg of body wt, i.p.) and prepared as previously described [5]. Following tracheostomy, a jugular vein and femoral artery were cannulated

lar fluid by a process of nonionic diffusion. Although there have been extensive studies of the pathway of ammonia biosynthesis and its regulation (for review, see Ref. 1), little information is available concerning quantitative aspects of the sites of ammonia entry into the tubular fluid.

Received for publication December 5, 1979 and in revised form May 5, 1980 0085-2538/81/0019—0281 $01.40

© 1981 by the International Society of Nephrology 281

Sonnenberg et a!

282

for infusion, and for blood pressure measurement and sampling, respectively. The left kidney was immobilized in a plastic (Lucite) cup, the papilla tip was exposed by opening the ureter near its insertion into the kidney, and urine was collected by gentle suction through an ureteral catheter. A priming dose of Ringer's solution (0.5 ml/100 g of body wt) containing tritiated inulin (33 /LCilml) was administered, followed by constant infusion of the same solution throughout the experiment at 0.5 mlIlOO g of body wt per hour. After surgery was completed, a 40-mm equilibration period was allowed before urine and duct fluid samples were collected. Consecutive 20-mm urine collections were taken over the subsequent 2 hours; arterial blood samples (50 to 100 l) were obtained at the midpoint of each collection. Fine polyethylene catheters (O.D., 20 to 30 m) were used to sample duct fluid as previously described [5]. Whenever possible, samples were ob-

tamed consecutively in the same collecting duct system from near the papillary tip, at intermediate depth, and deep in the medulla. In each animal, 7 to 9 samples of fluid were taken by controlled suction sufficient to overcome tip resistance of the catheter.

stopped with an excess of 1 N hydrochloric acid. The resultant 14C-glutamic acid was separated from 14C-2-oxoglutaric acid by ion exchange chromatography before analysis in a liquid scintillation counter (Cheema-Dhadli et al [8]).

Statistical evaluation of data was done by Student's paired and unpaired t test and regression analysis. Both linear and semilogarithmic plots were tested in the latter, and the one showing the higher correlation coefficient was chosen. For graphic representation, a correction factor was applied [9]. Results

Average body weights of rats placed on the low and high protein diets were 281 7 and 282 8 g, respectively (mean sEM). After 2 weeks on the regimen, the former weighed 273 8 g; and the latter, 304 8 g (P < 0.05). The weight gain of high protein rats was less than that expected for animals on a normal ad lib diet (approximately 35 glweek).

Average blood and plasma values for the two

The depth of insertion was measured with a mi-

groups at the time of experiment are shown in Table 1. There was no significant difference in hematocrit

crometer during withdrawal and was related to med-

or in electrolyte concentrations. Arterial blood

ullary length of the duct system obtained from a

pressures were higher (P <0.05), and plasma urea concentrations were lower (P < 0.01) on the low protein diet. The plasma urea concentration on the protein-enriched diet was, however, not different from that observed in a group of normal animals

postmortem saggital section of the kidney.

Concentrations of tritiated inulin in plasma,

urine, and duct fluid were measured by liquid scintillation counting; sodium and potassium in plasma and urine, by flame photometry; and chloride, by electrometric titration. Plasma urea concentration

was determined as described by Wybenga, Digiorgio, and Pileggi [6]; and urine ammomum, by the Formol titration method [7]. To measure the ammo-

nium concentration in 10-nl volumes of collecting duct samples, we first converted ammonium ions (NH4) plus ammonia (NH3) to radioactive gluta-

(6.8 mrvi

[SEMI 0.5) [10].

Renal functional data for the experimental kidney are given in Table 2. With the exception of potassium excretion, all values were somewhat lower in

the low-protein-fed rats. None of the differences were statistically significant, however. Urine am-

mate in the presence of 14C-2-oxoglutarate, NADH,

monium excretion was not significantly higher in rats on a high protein diet, probably because the method of pair feeding with low-protein-fed animals did not

glutamate dehydrogenase (ammonia-free), and adenosine diphosphate (ADP). This reaction was

result in normal weight gain or elevation of plasma urea concentration in the high protein diet group.

Table 1. Average blood and plasma data of rats on low and high protein dietsa

BP

Hct

mm Hg

%

Low protein(N= 5)

l28i

49

139

4.6

113

4.1C

Highprotein(N=5)

118

50

140

4.3

111

6.4

Values are the means p < 0.05, difference between groups. F < 0.01, difference between groups. SEM.

PNa

PK

Pci

Purea

mM

mEqiliter

283

Sites of ammonia addition 0.90

0.50

0.80

0.40

0 U-

0.70 0.301—

F-

I1

0.60 0.201—

0.50 U-

+

I-

0.10 F—

z

0.40

Tip

Base

0.30

Fig. 1. Paired collections in individual duct systems of rats fed low protein diet. Base collections were near the corticomedullary

border; tip collections were near the orifice. Average values

0.20

SEM are indicated by open circles.

0.10

The urine pH was not measured for the experimental kidney, but the urine pH for the contralateral kidney was 6.0 0.3 in the low-protein-fed rats and 6.3 0.1 in the high-protein-fed rats. To evaluate the content of ammonium along the collecting duct, we divided intratubular concentrations of ammonium by fluid-to-plasma ratios of inulin to correct for fluid reabsorption. The data from

Fig. 2. Paired collections in individual duct systems of high protein rats. Explanations are given in Fig. 1.

consecutive shallow (near the tip) and deep (near the corticomedullary junction) collections in the same duct system were paired and analyzed to determine whether net addition of ammonia to duct fluid could be demonstrated. Results of 15 such

tween deep and shallow sampling sites demonstrated net addition of ammonia to the medullary duct fluid. The mean values for deep and shallow

0 Tip

Base

mals are shown in Fig. 2. In 14 sets of collections, the statistically significant increase (P < 0.01) be-

in Fig. 1. A significant difference (P < 0.01) be-

collections were 0.29 0.06 and 0.50 0.05, respectively, and the average depths of insertion were 6.66 0.21 mm and 0.58 0.08 mm.

tween deep (base) and shallow (tip) collection sites was found, demonstrating net addition of ammonia along the course of the medullary duct. The mean values for deep and shallow collections were 0.18 0.03 and 0.29 0.03, respectively, and the average depth of insertion of the sampling catheter was 5.34 0.37 and 0.76 0.12 mm, respectively (mean SEM). Similar paired data in high-protein-fed ani-

To elucidate the pattern of transport along the duct system, we plotted all collections against the percent of medullary length. The increase in inulin concentration from corticomedullary border to papila tip in rats on both low and high protein intakes shows the expected fluid reabsorption (Fig. 3). The increased slope of the line in the low protein group is compatible with the relative reduction in urine

paired collections in low-protein-fed rats are shown

Table 2. Average renal function in experimental kidneys of rats on low and high protein dietsa

GFR

V

mi/mm

p2/mm

Low protein (N = 5)

1.04

3.96

Highprotein(N= 5)

1.08

5.25

a Values

are the means

UnV

UNaV

UKV

nEqlmin

nEq/min

UciV nEqimin

nEq/min

129

435

425

331

345

293

476

395

SEM. All data are given per gram of kidney weight.

Sonnenberg et a!

284 400

.

200

....

100

40

.

A

-

0

.

100r- A 40F-

.. 'S

20 I—

. ••

..

10

10

LL

I400

L

__lU

I

I

4

I..

.

B

•

200

..

100

'

.•

I

U 200

B

100

40

20—

I

0

40

20

I 60

Length, %

I

80

lU

100

0.82, P < 0.01.

in part to fluid reabsorption. But, when the data were corrected for this factor (Fig. 5), a statistically

significant addition of ammonia was present throughout the medullary collecting duct system. To obtain a quantitative estimate of the amount of ammonia added in this nephron segment, we multiplied the paired deep and shallow determinations for ammonium concentration, divided by the corresponding (TF/P)1, by the GFR in each rat, and we obtained the means for the results. In rats fed a low protein diet, the absolute ammonium load increased from 175 25 to 281 36 nEq/minig of kidney wt.

In rats fed a high protein diet, the corresponding 59 nEq/minlg of

Discussion

4

..•

.

10

••

4

2—

flow (Table 2). The concentration of ammonium in duct fluid is shown in Fig. 4. In both groups, there was a steep increase from cortex to papila tip, due

61 and 544

....

Urine

(A)1ny=0.025x+3.O3;r=0.74,P<0.0l.(B)lny0.0l8x+

values were 314 kidney wt.

•

40 20

Fig. 3. Change of inulin concentration along the medullary collecting system of A rats fed low protein and B ratsfed high protein diets. Calculated regression lines are indicated by solid lines:

3.48;

S.

....

20

0.

•• • . .

• I 0

I

I

I

I

20

40

60

80

Length, %

lU

100

Urine

Fig. 4. Change in ammonium concentration along the medullary collecting system of(A) rats fed low protein and (B) rats fed high = 0.77, P < 0.01. (B) protein diets. (A): ln y = 0.038x + 0.65; = 0.85, P < 0.01. ln y = 0.029x + 1.73;

nine. They suggest that ammonia enters the tubular fluid from the blood downstream from the glomeru-

lus and distributes itself by nonionic diffusion as dictated by the hydrogen ion concentration in the various compartments. About this same time, Gottschalk, Lassiter, and Mylle [12] reported that the pH of the tubular fluid increased as fluid descended into the ioop of Henle. This alkalinization should raise the plasma ammonia in this nephron segment and favor the diffusion of ammonia from the loop of Henle to the collecting duct. According to this con-

cept, water abstraction, which occurs in the descending limb of Henle's loop, concentrates the

Balagura and Pitts [11] have shown that when ammonia and creatinine are injected into the renal ar-

tubular fluid bicarbonate more than carbon dioxide

tery, ammonia arrives in the urine prior to creati-

pH displaces the steady state of the reaction,

does and causes a rise in the pH. This rise in the

Sites of ammonia addition 0.4

S. •• .

A

o

285

golden hamster but did not provide quantitative information. The available micropuncture data are conflicting. Hayes et al [3] measured the ammonium and inulin concentrations in the lumen of superficial proximal and distal tubules, as well as in urine

during mannitol diuresis, and calculated that if these values could be applied to all nephrons, there was an adequate quantity of ammonium in the dis-

tal tubular fluid to account for all of the urinary ammonium. On this basis, they suggested that there was no significant addition of ammonia directly to collecting duct fluid, but the diuretic conditions of these experiments might be important in decreasing

::

• .

•.

0.10

ammonia addition to the collecting duct. By contrast, Glabman, Kiose, and Giebisch [4] reported that, in sodium-loaded rats, ammonia was added between the distal tubule and the final urine. But in most samples, the inulin concentration was not measured. In the few samples where both inulin

0.04

and ammonium were determined, ammonia addition appeared to have occurred both in the distal tubule and the collecting duct, but the data were not actu-

0.02

ally presented. These micropuncture results are less than definitive because of possible nephron

0

I

I

20

40

I 60

I

80

Length, %

lU

100

Urine

Fig. S. Intratubular ammonium corrected for fluid reabsorption

in the medullary duct of (A) ratsfed low protein and (B) rats fed 0.41, P <0.01. high protein diets. (A): in y = 0.OlOx — 2.25;

(B)InyO.Ollx— 1.81;r=0.56,P<0.01.

heterogeneity with varying contributions of ammonium to the final urine made by the superficial and

deep nephrons. In addition, they do not examine ammonia handling in the medullary as distinct from the cortical segment of the collecting duct.

Our interest in the intrarenal handling of ammonia was stimulated initially by a serendipitous observation that the oral administration of sodium

bicarbonate to patients with the ketoacidosis of NH4 NH3 + W, to the right, thereby increasing

fasting caused a significant decrease in the urine pH

the plasma ammonia at the papillary tip (water abstraction in excess of ammonia will also increase the plasma ammonia). The resultant high plasma ammonia concentration produces a favorable diffusion gradient for the transfer of ammonia from the ioop of Henle into the collecting duct. Further evidence for this hypothesis was obtained by Robinson and Owen [13], who noted that during antidiuresis, the concentration of total ammonia in the renal tissue water rose progressively from the cortex to the tip of the papilla, and also by DuBose et a! [14], who confirmed that the pH of lumenal fluid in the ioop of Henle was higher than that in the renal cortex.

rather than the expected rise. Subsequently, we

Although indirect evidence suggests that ammonia may be added to the collecting duct system, direct examination of this issue has been limited. Ullrich, Huger, and KlUmper [2], using the microcatheterization technique, reported that ammonia was added to the medullary collecting duct of the

documented that small i.v. loads of sodium lactate produced the same result, and that the ammonium excretion decreased, contrary to the predictions of the nonionic diffusion hypothesis [11]. On the basis of this observation, we suggested that the "paradoxical" aciduria of fasting was due to decreased transfer of ammonia from the loop of Henle to the collecting duct [15]. Subsequent studies in fasting patients with chronic metabolic acidosis demonstrated that the urine pH correlated directly with the urine ammonium rather than inversely as predicted by the nonionic diffusion hypothesis [16]. These data and those of Tannen [17] indicated that the urinary ammonium was an important determinant of the urine pH. This finding suggested that a significant quantity of ammonia was added to the collecting duct, because ammonia addition to tubular fluid would only affect the pH in a segment where

286

Sonnenberg

et a!

the secretion of hydrogen ions was the primary

reported in this paper. First, because it is well

factor limiting the final urine pH (that is, a segment where hydrogen ion secretion was "secretory lim-

known that ammonium excretion is generally higher in urine with a lower pH, and that this major decline in pH occurs between the surface distal tubules and final urine [14, 18, 22], it follows that the bulk of the new ammonia added during urine acidification probably occurs in the collecting duct (providing that the quantity required exceeds that already in the lumena] fluid). Second, if about 40% of ammonium excretion results from ammonia entry into the medullary collecting duct, it follows that there was also a commensurate addition of hydrogen ions, or the tubular fluid pH would rise. Because there is good evidence

ited"). In contrast, all other nephron segments appear to have a relatively high secretory capacity and normally manifest predominantly a gradientlimited type of secretory control (for review, see Ref. 18).

The results reported in this paper indicate that both the concentration and quantity of ammonium rise as fluid traverses the medullary collecting duct. The rise in ammonium concentration in part reflects water abstraction, but also net addition of ammonium into the collecting duct fluid (Figs. 1, 2, and 5).

In quantitative terms, the amount of ammonia added in the medullary collecting duct, when estimated by the ammonium-to-(TFIP)1 ratio multiplied by 68 the individual GFR, was 106 32 and 231 nEq/minlg of kidney in the rats fed a low and high protein diet, respectively (see Results section). The actual values are only approximate because there were no timed quantitative collections of medullary collecting duct fluid in these experiments. Nevertheless, this quantity represents about 40% of the excreted ammonium in the rats fed the low and the high protein diet (Table 2). In these studies, nephron functional heterogeneity does not influence the results because the distal tubules of both deep and superficial nephrons join

the collecting duct in the renal cortex before the first (deep) site of sampling of medullary collecting duct fluid [19, 20]. The results reported in this paper

in rats are consistent with the earlier microcatheterization studies of Ullrich, Huger, and Klümper [2] in the golden hamster and, furthermore, add a quantitative dimension.

Whether the ammonia added to the medullary collecting duct fluid was produced by the cells in this region, or whether it was produced in the cortex and transferred from the loop of Henle to the collecting duct, or both, cannot be ascertained from our studies. The enzymes required for the production of ammonia are present throughout the entire nephron, but are highest in concentration in the proximal tubule. Furthermore, Curthoys and Lowry [21] have shown that phosphate-dependent glutaminase does not show an adaptive increase in the medulla, whereas it does in the proximal tubule dur-

ing metabolic acidosis. Thus, it seems likely that most of the urinary ammonia is produced in the cortex, although a significant contribution of the medullary tissue cannot be excluded. Several implications follow from the observations

that pH decreases along the collecting duct [18], this nephron segment probably secretes a considerable amount of hydrogen ions under these conditions. Third, the potential importance of a transfer of ammonia from the loop of Henle to the collecting

duct can be appreciated if we consider that in its absence about half of the ammonia added to the proximal tubular fluid would, by necessity, be lost from the tubular fluid in the most distal portion of the distal convoluted tubule. This follows from the following three facts (the first two are from superficial nephrons only): (1) the pH of the proximal exceeds that of the distal tubular fluid by about 0.3 pH units [22]; (2) the tubular fluid volume is reduced by approximately fourfold as evidenced by the (TFIP)1

ratios in the end-proximal and end-distal convoluted tubules [23]; and (3) the plasma ammonia concentration is in diffusion equilibrium throughout the renal cortex. Thus, the ammonium concentration should be higher (twofold) in end-distal convoluted tubule, but the relatively greater fall in the end-distal tubular fluid volume implies that the total ammonia content will be less in the distal vs. the proximal tubule. The study of Finkelstein and Hayslett [24], demonstrating that papillectomy significantly impairs the ability to excrete ammonium, offers support for the concept that the medullary structures play an important role in ammonium excretion. These investigators compared the ability of papillectomized animals to acidify the urine and excrete ammonium to that of partially nephrectomized controls with a

similar GFR. They observed that although both groups of animals could acidify the urine normally, the papillectomized group excreted 50% less ammonium. These studies demonstrating a significant limitation of papillectomized animals to excrete ammonium and our findings that about 40% of the urinary

ammonium is added to the medullary collecting duct in rats on a low or high protein diet suggest an

Sites of ammonia addition

important role for the renal medullary structures in ammonia excretion in the rat. Acknowledgment

A portion of this study has been published in abstract form (Kidney mt 16:837, 1979). These studies

were supported by grants from the Medical Research Council of Canada (MRC MT2836, MT4043

and MA5623), and the St. Michael's Hospital Research Society. B. Gardner, C-B. Chen, S-C. Tam, R. Hamat, K. Sonnenberg, U. Honrath, A.T. Yeress, C.K. Chong, and B. Halperin gave technical assistance. Reprint requests to Dr. M.L. Ha/penn, Lab #1, Research Wing, Si. Michael's Hospital, 38 Shuter Street Annex, Toronto, Ontario, M5B 1A6, Canada

References 1. TANNEN RL: Ammonia metabolism. Am J Physiol 235:F265—277, 1978 2. ULLRICH KJ, HILGER HH, KLUMPER JD: Sekretion von Am-

moniumionen in den Sammelrohren der Saugetierniere. Pflueger's Arch 267:244—250, 1958 3. HAYES CP JR, MAYSON JS, OWEN EE, RoBINsoN RR: A

micropuncture evaluation of renal ammonia excretion in the rat. Am J Physiol 207:77-83, 1964 4. GLABMAN S, KLOSE RM, GIEBISCH G: Micropuncture study

of ammonia excretion in the rat. Am J Physiol 205:127—132, 1963

5. SONNENBERG H: Medullary collecting duct function in anti-

diuretic and in salt or water diuretic rats. Am J Physiol 226:501-506, 1974 6. WYBENGA D, DJGI0RGI0 J, PILEGGI VJ: Manual and auto-

mated methods for urea nitrogen measurement in whole serum. C/in Chem 17:891—895, 1971

7. CHAN JCM: The rapid determination of urinary titratable

287

10. WILSON DR, SONNENBERG H: Medullary collecting duct function in the remnant kidney before and after volume expansion. Kidney mt 15:487-501, 1979 11. BALAGURA S, PITTS RF: Excretion of ammonia injected into a renal artery. Am J Physiol 203:11-14, 1962 12. GOTTSCHALK CW, LASSITER WE, MYLLE M: Localization

of urine acidification in the mammalian kidney. Am JPhysiol 188:581—585, 1960

13. RoBINsoN RR, OWEN EE: Intrarenal distribution of am-

monia during diuresis and antidiuresis. Am J Physiol 208:1129—1134, 1965 14. DUBOSE TD, HOGG RJ, PUCACCO LR, LuccI MS, CARTER

NW, KOKKO JP: Direct determination of pH and PCO2 in

cortical and medullary nephron segments of the Munich Wistar rat. C/in Res 27:4l3A, 1979 15. STINEBAUGH BJ, MARLISS EB, GOLDSTEIN MB, Fox IH, SCHLOEDER FX, HALPERIN ML: Mechanism for the para-

doxical aciduria following alkali administration to prolonged-fasted patients. Metabolism 24:915—922, 1975 16. SCHLOEDER FX, STINEBAUGH BJ: Urinary ammonia content

as a determinant of urine pH during chronic metabolic acidosis. Metabolism 26:1321—1331, 1977

17. TANNEN RL: The response of normal subjects to the short ammonium chloride loading test: the modifying influence of renal ammonia production. C/in Sci 41:583—595, 1971 18. RECTOR FC JR: Micropuncture studies on the mechanism of urine acidification, in Renal Metabolism and Epidemiology of Some Renal Diseases, edited by METCOFF J, York, Pennsylvania, The Maple Press Company of York, 1974, pp. 931

19. KRIZ W: Der architektonische und funktionelle Aufbau der Ratteniere. Z Zellforsch Mikrosk Anat 82:495-535, 1967 20. OLIVER J: Nephrons and Kidneys. New York, Hoeber Medical Division of Harper and Row, 1968, p. 104 21. CURTHOYS NP, LOWRY OH: The distribution of glutaminase

isoenzymes in the various structures of the nephron in normal, acidotic and alkalotic rat kindey. J Biol Chem 248:162168, 1973

22. MALNIC G, DEL MELLO AIRES M: Micropuncture study of

renal hydrogen ion transport in the rat. Am J Physiol

acid and ammonium and evalution of freezing as a method of preservation. Clin Biochem 5:94-98, 1972

23. SONNENBERG H: Proximal and distal tubular function in salt-

8. CHEEMA-DHADL] S. IJAMAT R, SONNENBERG H, HALPERIN

deprived and salt-loaded desoxycorticosterone acetate es-

ML: A micromethod to measure ammonia. Kidney mt

caped rats. J Clin Invest 52:263—272, 1973 24. FINKELSTEIN FO, HAYSLETT JP: Role of medullary structures in the functional adaptation of renal insufficiency. Kidney mt 6:419—425, 1974

19:80—82, 1981

9. BRACE RA: Fitting straight lines to experimental data. Am J Physiol 233:R94-99, 1977

222:147—158, 1972