AMERICAN JOURNAL OF PHYSIOLOOY Vol. 229, No. 5, November 1975. Prinkd
Distal renal
in U.S.A.
tubular
tracer
tubular
microinjection
potassium
study
transport
N. FOWLER, G. GIEBISCH, AND G. WHITTEMBURY Department of Physiology, Yale University School of Medicine, New Haven, Connecticut Bioquimica, lnstituto Venecolano de Investigaciones Cientijcas, Caracas, Venezuela
N., G. GIEBISCH, AND G. WHITTEMBURY. microinjection study of renal tubutar potassium
FOWLER,
tracer
Distal tubular transport. Am. J.
Physiol. 229(5) : 1227-l 233. 1975.-Renal tubular potassium (K) transfer was studied in rats using a tracer microinjection technique in which [W]inulin and 42K were simultaneously injected into early distal tubules during osmotic diuresis. Experiments were carried out in 1) animals on a control diet, 2) animals in which K secretion had been stimulated (high-K diet + KCl, NaySO+ Diamox infusions), and 3) animals in which K excretion had been reduced by a low-K or low-Na diet or by amiloride. 42K excretion into the urine coincided closely in time with the excretion pattern of [‘“cl inulin. Efflux of 42K out of the lumen was stimulated during reduced K secretion along the distal nephron and decreased during enhanced K secretion when small tubular K loads were given. These experiments demonstrate bidirectional K movement across the distal nephron and show that changes in reabsorptive K efflux participate in the regulation of tubular net K movement. distal tubule; collecting duct; 42K efflux; tubular bidirectional K movement
K reabsorption;
OBSERVATIONS SUPPORT the view that the distal tubule and the collecting duct are the nephron sites where important alterations in potassium transport occur (3, 11, 12, 17, 18, 20, 2 1, 22, 24, 26, 32) These studies in vivo on single distal tubules and on collecting ducts have demonstrated that the mechanism of potassium transport residing at these nephron sites responds to a variety of metabolic stimuli and that both net secretion as well as net reabsorption of potassium may occur. In the present study some aspects of the potassium transport system were further studied by intratubular tracer injection methods (16, 28). Experiments were carried out on distal tubules utilizing &K. From the difference between the amount of tracer injected and recovered in the urine, an estimate may be obtained of efflux of labeled potassium from the distal tubule and the collecting duct, providing information on the egress of potassium from the tubular lumen under different experimental conditions. Evidence is available that bidirectional movement of potassium ions takes place across distal tubule cells and that unidirectional flux components respond to a variety of stimuli (8). It can further be inferred from micropuncture and microperfusion studies on distal tubules that potassium efflux from the lumen is mediated by both passive and active MANY
l
of
0651U;
and Centro
de Biojkica
y
pathways (10, 12, 15, 22, 23, 30). Although separation into these flux components was not carried out in the present study, we have observed that stimulation of tubular potassium secretion can be associated with reduced loss of injected tracer from the lumen, whereas curtailment of tubular potassium secretion is uniformly accompanied by a significantly enhanced rate of 42K loss from the tubular lumen. These findings underscore the role played by changes in potassium efflux from the lumen in modulating net transport rate of potassium at nephron sites beyond the early distal tubule. METHODS
Experiments were performed on Long-Evans rats weighing 180-270 g and anesthetized with Inactin, 100 mg/kg. The methods of preparing the animals for micropuncture, the catheterization of the jugular veins, carotid artery, and the ureter, and the exposure of the kidney have been described in previous papers (10, 18, 24). To provide high urine flow rates necessary for rapid serial collections of urine, most animals were made diuretic by the intravenous infusion of a 2.5 % NaCl solution at a rate of 3.2 ml h-l ‘100 g-l body wt. Only in the low-sodium rats (see below) 2 % mannitol was administered intravenously at a rate of 1.6 mlh-l 100 g-l body wt. This was done to maintain the state of reduced urinary sodium concentration in those animals that had been kept on the low-sodium diet. After an equilibration period of at least 45 min, several 20-min urine collection periods from the experimental kidney were used for the measurement of glomerular filtration rate (chemical inulin determinations by the method of Vurek and Pegram (31)) and urinary excretion rates of sodium and potassium by flame photometry (Instrumentation Laboratory, Inc., Boston, Mass.). Sodium and potassium excretion rates were expressed as fractions of the filtered load. The following experimental groups of rats were studied: I) control rats kept on a diet of Purina laboratory chow (0.72 % K, 0.46 % Na) until the night preceding the experiment, having free access to water. 2) Rats kept on a highpotassium intake (General Biochemicals, 15 g K/kg diet) for several weeks prior to the experiment. In this group of animals, a high urinary excretion rate of potassium during the microinjection experiment was achieved by: a) the addition of enough KC1 to the 2.5 % NaCl solution to l
l
1227
Downloaded from www.physiology.org/journal/ajplegacy at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
1228
FOWLER,
achieve a final concentration of 60 meq/liter b) by giving the carbonic anhydrase inhibitor Diamox at a rate of 40 mg h-1 kg- l body wt; and c) by giving a 2 X isosmotic, equimolar mixture of sodium sulfate and potassium chloride. 3) Animals kept on a low-potassium diet (General Biochemicals) for several weeks prior to the experiment. 4) Animals kept on a low-sodium diet (General Biochemicals) for several weeks prior to the experiment. 5) Rats kept on a control diet receiving amiloride at a rate of 2.4 mg kg-i h-l. These different regimes resulted in a wide range of urinary excretion rates of potassium and result, as shown in other micropuncture experiments, in greatly differing secretory transport patterns across the distal tubular epithelium (11, 17, 20-24, 32). A stock solution of isotonic NaCl containing 100 pCi/ml of [14C]inulin (New England Nuclear Corp., Boston, Mass.) was used to dilute a high specific activity 42KC1 solution (New England Nuclear Corp., about 5 &i/ml at time of shipment) to achieve a calculated final potassium concentration of 5-l 5 meq/liter. These concentrations and isosmolarity were verified by macroflame photometry and osmometry measurements. A small amount of lissamine green (500 nl of a 10 % solution were added to 35 ~1 of injectate) was used to color the microdroplets. Measurement of the radioactivity of known volumes of injectate by the use of calibrated constriction pipettes (3, 10, or 30 nl) in a Mark I Liquid scintillation counter (Nuclear-Chicago Corp.) allowed for the measurement of the total amounts of radioactivity due to [14C]inulin and 42K injected. Isotope separation and overlap corrections between appropriately set counting windows were accomplished by the channel ratio method, applying appropriate corrections for *K decay. Assuming complete excretion of inulin, tubular efflux of 42K was calculated as the difference between injected and excreted moieties. Microinjection sites were selected for early distal localization by the administration of small amounts (0.04-0.06 ml) of a 5 % lissamine green solution (33), and care was taken to avoid retrograde flow during the injection of the isotope mixture. The magnitude of the injected sample was 3, 10, and 30 nl in high-K, low-sodium, and all other experimental conditions, respectively, and the mean duration of injections was 30 s (range 15-45 s). Urine was collected from the left ureter into counting vials containing 10 ml of Aquasol (New England Nuclear Corp., Boston, Mass.). Immediately following the injection, collections were made every 15 s for the first 2 min and every 30 s for an additional time period of 3 min. The mean inulin recovery of all injections was 96 %, and samples were not accepted if the total cumulative inulin recovery did not exceed 94 70. Data are expressed as mean values rf: standard error, and the Student t test was used to assess the statistical significance of differences. l
l
l
GIEBISCH,
AND
WHITTEMBURY
TABLE 1. Intratubular injections of [42K] and [‘4C]inulin---summary of renal excretion data _-____ .-~~-~-- ~ ~ -~ _-~---.~-~~~ _ ~_ .- -- __.-.--~~-_-.Exptl
Condition
l_____--l_. Control
I
High
K
High
K + Diamox
n
- --
Urine Flow, ml/min +kg __----~~
FE
- ----
Ns,
Na/In U/P _.-_-__
FEK, K/In U/P .---.-
---~
16
0.335~0.009
16
0.457&0.016
0.8837hO.0298
8
0.475jzO.016
1.510
jzO.0890
1.113
&0.0080
0.1075&0.0028
0.3220
~0 .0125
l
RESULTS
A summary of renal excretion data, including urine flow rates and fractional excretion rates of sodium and potassium, is represented in Tables 1, 2, and 3. Table 1 shows the results of experiments of those animals receiving a total tubular volume of injection of 30 nl. It is apparent that the
+
NCi2SO4
Low
K
KCI
12 32
0.375zt0.011
0.0800=t0.0013
highest urine flow rates were observed during stimulation of potassium excretion, achieved by the intravenous administration of an exogenous potassium load, by Diamox or sodium sulfate. As expected from the infusion of hypertonic sodium-containing solutions, the rate of fractional urinary sodium excretion is uniformly increased to levels between 8 and 15%. In contrast, fractional excretion rates of potassium vary and depend strongly on the experimental condition. Compared to control animals excreting an amount equivalent to some 32 %’ of the filtered potassium load, urinary potassium excretion is dramatically augmented in those groups of animals pretreated with a high-potassium diet and receiving potassium-containing solutions intravenously during the microinjection experiments. On the other hand, it is apparent that both a low-potassium diet or the administration of amiloride sharply reduced fractional urinary excretion rates to about one-fourth of the control values. Table 2 contains similar data on urine flow rates and fractional sodium and potassium excretion rates in control rats which, instead of 2.5 %I NaCl, received 2 %j mannitol at a rate of 1.6 ml . h-l -100 g-l. This infusion was chosen because in the rat it does not produce a marked natriuresis, and hence it allows for the demonstration of the effects of a low-sodium diet upon urinary sodium and potassium excretion rates. An injection volume of 10 nl was chosen, and the sodium concentration of the tubular injectate reduced to 10 meq/liter to avoid overloading of the tubular sodium transport mechanism. It has been demonstrated that dietary pretreatment with a low-sodium diet reduces both sodium and potassium excretion rates (11, 22). This was fully confirmed in the present study. Inspection of Table 2 indicates that in low-sodium animals the fractional sodium excretion rate drops to less than $&th of the control sodium excretion rate. Similarly, fractional potassium excretion rates are reduced to about one-sixth of control excretion rates. Table 3 summarizes data from a series of experiments similar to those summarized in Table 1, with the exception that in the control and high-K animals summarized in Table 3, the tubular rate of injection was lower by a factor of 10, i.e., 3 instead of 30 nl. It is apparent that fractional urinary excretion rates of potassium were greatly augmented above the level of excretion rate achieved under control conditions by the pretreatment with a high-K diet and the administration of the potassium-containing infusion fluids.
Downloaded from www.physiology.org/journal/ajplegacy at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
RENAL
TUBULAR
1229
K TRANSPORT
2. Intratubutar injections of [42K] and [W]z’nuhsummaryof renal excretion data
3Uni
TABLE
Exptl Condition
Urine Flow, ml/min * kg
s
ConrYoz 2
12
Low
20
Na
0.087&0.0058 035 *o. 0005
0
l
FEK,
FEN*,
&/In
60 -
U/P
K/In
0.02590~0.00150 0.00021*0.00002
U/P
0.3170~0.0013 0.0537&0.0028
VaIucs are means & SE. n, number of clearance periods. Animals received 2% mannitol at a rate of 1.6 ml/h. 100-g ralt. Injected volume, 10 nl. FEN,, fractional excretion ot wxlium; FEK, fractional excretion of potassium.
Conirol High
tL 3
K
u$e;1g.
FEN&,
Na/In
50 -
50 -
50
40 -
40 I-
40
30 -
30 -
30
z kz $
20 -
20 -
20
IO 1
IO -
IO
FEK, U/P
12
0.370~0.018
0.124&0.0047
0.250&0.0004
8
0.230&0.011
0 * 079 ho * 0003
0.650&0.0300
Vaiues are means & SE. n, number of clearance periods. NaCl at a rate of 3.2 ml/h* 100-g rat. Injected volume, 3 nl. cretion of sodium; FEK, fractional excretion of potassium.
100
R/In
U/P
Animals received FEN,, fractional
60 -
2I :: zz Y
TABLE 3. fntratubular injectionsof L4”K] and [14C]inulinsummaryof renal excretiondata
Exptl Condition
& Y
80
80
Y e
60
60
40
40
20
20
0
2.5% ex-
? s -I 2 z ae
Figures l-4 show representative examples of microinjection data obtained in the different control conditions and during various states of enhanced or reduced urinary potassium excretion rates. Table 4 summarizes mean recovery rates of 42K corrected for inulin loss (always less than 6 %) in the different experimental conditions. Figure 1 provides information on the excretion patterns of [%]inulin and 42K after early distal tubular injections in control animals. The top graph depicts fractional recovery rates, and the lower graphs depict total cumulative recoveries. Tubular microinjections were made at a rate of 3, 10, and 30 nl, respectively. It is apparent that excretion of [14C]inulin was complete within 3-4 min. Three points deserve mention. First, the excretion of both isotopes was somewhat delayed, although still reaching constant levels within 3-4 min in the control experiment in which an intravenous infusion of mannitol (2 % at 1.6 ml h-l - 100 g body wt-‘) was given instead of saline (2.5 %, NaCl at 3.2 ml 4h-l 100 g body wt-l). This was due no doubt to the lower urine flow rates of the animals receiving mannitol. (Compare mean urine flow rates in Tables 1, 2, and 3.) This indicates that reproducible excretion patterns obtain over the range of urine flow rates achieved in the present series of experiments. A second point concerns the total rate of 42K excretion rates in the three representative control experiments. It can be seen that the recovery of 42K is inversely proportional to the total volume, i.e., amount of l12K injected. Inspection of Table 4 shows that the mean ratio of excretion of 42K/[14C]inulin was 85.9, 74.9, and 62.0 % in the animals receiving 30-, lo-, and 3-nl injections. These differences are statistically significant (P < 0.01 : control experiments in which 30 and 10 nl were given; P < 0.005: control experiments in which 30 and 3 nl were given; P < 0.05 : control experiments in which 10 and 3 nl were given). Finally, the similarity of the inulin and potassium excretion patterns should be noticed. With respect to [lAC]inulin, the 42K recovery is only slightly delayed and in general, the excretion patterns of potassium follow closely that of inulin. A similar excretion pattern was also found by De Roufignac and Guinnebault (9). Thus, as defined by Gottschalk, l
80 60
I
40
20
t
0
0
I
0
MIN
l
3nI
IUnl
MIN
2
-.I
3
4
MIN
FIG. 1. Comparison of [14C]inulin and 4% excretion patterns after early distal tubular microinjections in rats on a control diet. TOP, Bottom. fractional recovery. Shaded area : 42K, open area: [14C]inulin. cumulative recovery. Open circles: [14C]inulin, filled circles: 42K reintravenous infusion: 2.5y0 NaCl at a rate of coveries. Left panel. injection volume: 30 nl. 3.2 ml-h- ‘400 g body wt- l. Intratubular Middle panel: intravenous infusion: 2% mannitol at 1.6 ml. h-l* 100 g body wt? Intratubular injection volume: 10 nl. Right panel: intravenous infusion: 2.5y0 NaCl at a rate of 3.2 ml h-l 100 gm body wt? Intratubular injection volume: 3 nl. l
l
Morel, and their associates (16, 28), potassium recovery is LCdirect,” i.e., with a time course similar to that of the simultaneously injected inulin. Figure 2 summarizes examples of the time course of [‘4C]inulin and 42K recovery in animals in which a total volume of 30 nl was injected into the early distal tubule and in which urinary potassium excretion rates diKered significantly from that in control rats. Compared to the control excretion patterns of inulin and potassium (see Fig. 1, left panel), both the time course and the extent of excretion of 42K was unchanged in animals in which potassium excretion was stimulated (high-K animals), whereas total recovery of injected 42K was significantly reduced, although not delayed, in animals deprived of dietary potassium lor several weeks (low-K animals) and in animals in which the rate of urinary potassium excretion had been curtailed by the prior administration of amiloride, a mildly diuretic agent known to inhibit potassium secretion at the distal tubular level (10). Inspection of Table 4 shows that the mean 42K/[14C]inulin ratios are similar in control I and high-K animals, whereas these ratios are significantly depressed in low-K and amiloride-treated animals (P < 0.001). Figure 3 compares 14C and 42K excretion patterns in animals receiving 2 % mannitol intravenously to stimulate urine flow rate. Sodium excretion rate was varied by dietary sodium withdrawal. On the left, a representative control excretion patterns is shown; on the right the recovery pattern of an experiment in which pretreatment with a lowsodium diet had led to significant reduction of both urinary sodium and potassium excretion. It is apparent that the 42K recovery is reduced in the low-sodium animals. The respec-
Downloaded from www.physiology.org/journal/ajplegacy at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
1230
FOWLER,
4. Recovery of microinjected [42K] compared to [‘qC]inulin excretion in diyerent experimental condz’tions -_I~-~
CONTROL
% 4$jjg$”
?z
I (30 nl)
85.93&2.42+
12
High
K (30 nl)
88.98kO.98
13
NW
High
K
82.18jz2.66
9
NSt
78.85A5.04
11
NW
55.96h5.40
14
Distal renal
in U.S.A.
tubular
tracer
tubular
microinjection
potassium
study
transport
N. FOWLER, G. GIEBISCH, AND G. WHITTEMBURY Department of Physiology, Yale University School of Medicine, New Haven, Connecticut Bioquimica, lnstituto Venecolano de Investigaciones Cientijcas, Caracas, Venezuela
N., G. GIEBISCH, AND G. WHITTEMBURY. microinjection study of renal tubutar potassium
FOWLER,
tracer
Distal tubular transport. Am. J.
Physiol. 229(5) : 1227-l 233. 1975.-Renal tubular potassium (K) transfer was studied in rats using a tracer microinjection technique in which [W]inulin and 42K were simultaneously injected into early distal tubules during osmotic diuresis. Experiments were carried out in 1) animals on a control diet, 2) animals in which K secretion had been stimulated (high-K diet + KCl, NaySO+ Diamox infusions), and 3) animals in which K excretion had been reduced by a low-K or low-Na diet or by amiloride. 42K excretion into the urine coincided closely in time with the excretion pattern of [‘“cl inulin. Efflux of 42K out of the lumen was stimulated during reduced K secretion along the distal nephron and decreased during enhanced K secretion when small tubular K loads were given. These experiments demonstrate bidirectional K movement across the distal nephron and show that changes in reabsorptive K efflux participate in the regulation of tubular net K movement. distal tubule; collecting duct; 42K efflux; tubular bidirectional K movement
K reabsorption;
OBSERVATIONS SUPPORT the view that the distal tubule and the collecting duct are the nephron sites where important alterations in potassium transport occur (3, 11, 12, 17, 18, 20, 2 1, 22, 24, 26, 32) These studies in vivo on single distal tubules and on collecting ducts have demonstrated that the mechanism of potassium transport residing at these nephron sites responds to a variety of metabolic stimuli and that both net secretion as well as net reabsorption of potassium may occur. In the present study some aspects of the potassium transport system were further studied by intratubular tracer injection methods (16, 28). Experiments were carried out on distal tubules utilizing &K. From the difference between the amount of tracer injected and recovered in the urine, an estimate may be obtained of efflux of labeled potassium from the distal tubule and the collecting duct, providing information on the egress of potassium from the tubular lumen under different experimental conditions. Evidence is available that bidirectional movement of potassium ions takes place across distal tubule cells and that unidirectional flux components respond to a variety of stimuli (8). It can further be inferred from micropuncture and microperfusion studies on distal tubules that potassium efflux from the lumen is mediated by both passive and active MANY
l
of
0651U;
and Centro
de Biojkica
y
pathways (10, 12, 15, 22, 23, 30). Although separation into these flux components was not carried out in the present study, we have observed that stimulation of tubular potassium secretion can be associated with reduced loss of injected tracer from the lumen, whereas curtailment of tubular potassium secretion is uniformly accompanied by a significantly enhanced rate of 42K loss from the tubular lumen. These findings underscore the role played by changes in potassium efflux from the lumen in modulating net transport rate of potassium at nephron sites beyond the early distal tubule. METHODS
Experiments were performed on Long-Evans rats weighing 180-270 g and anesthetized with Inactin, 100 mg/kg. The methods of preparing the animals for micropuncture, the catheterization of the jugular veins, carotid artery, and the ureter, and the exposure of the kidney have been described in previous papers (10, 18, 24). To provide high urine flow rates necessary for rapid serial collections of urine, most animals were made diuretic by the intravenous infusion of a 2.5 % NaCl solution at a rate of 3.2 ml h-l ‘100 g-l body wt. Only in the low-sodium rats (see below) 2 % mannitol was administered intravenously at a rate of 1.6 mlh-l 100 g-l body wt. This was done to maintain the state of reduced urinary sodium concentration in those animals that had been kept on the low-sodium diet. After an equilibration period of at least 45 min, several 20-min urine collection periods from the experimental kidney were used for the measurement of glomerular filtration rate (chemical inulin determinations by the method of Vurek and Pegram (31)) and urinary excretion rates of sodium and potassium by flame photometry (Instrumentation Laboratory, Inc., Boston, Mass.). Sodium and potassium excretion rates were expressed as fractions of the filtered load. The following experimental groups of rats were studied: I) control rats kept on a diet of Purina laboratory chow (0.72 % K, 0.46 % Na) until the night preceding the experiment, having free access to water. 2) Rats kept on a highpotassium intake (General Biochemicals, 15 g K/kg diet) for several weeks prior to the experiment. In this group of animals, a high urinary excretion rate of potassium during the microinjection experiment was achieved by: a) the addition of enough KC1 to the 2.5 % NaCl solution to l
l
1227
Downloaded from www.physiology.org/journal/ajplegacy at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
1228
FOWLER,
achieve a final concentration of 60 meq/liter b) by giving the carbonic anhydrase inhibitor Diamox at a rate of 40 mg h-1 kg- l body wt; and c) by giving a 2 X isosmotic, equimolar mixture of sodium sulfate and potassium chloride. 3) Animals kept on a low-potassium diet (General Biochemicals) for several weeks prior to the experiment. 4) Animals kept on a low-sodium diet (General Biochemicals) for several weeks prior to the experiment. 5) Rats kept on a control diet receiving amiloride at a rate of 2.4 mg kg-i h-l. These different regimes resulted in a wide range of urinary excretion rates of potassium and result, as shown in other micropuncture experiments, in greatly differing secretory transport patterns across the distal tubular epithelium (11, 17, 20-24, 32). A stock solution of isotonic NaCl containing 100 pCi/ml of [14C]inulin (New England Nuclear Corp., Boston, Mass.) was used to dilute a high specific activity 42KC1 solution (New England Nuclear Corp., about 5 &i/ml at time of shipment) to achieve a calculated final potassium concentration of 5-l 5 meq/liter. These concentrations and isosmolarity were verified by macroflame photometry and osmometry measurements. A small amount of lissamine green (500 nl of a 10 % solution were added to 35 ~1 of injectate) was used to color the microdroplets. Measurement of the radioactivity of known volumes of injectate by the use of calibrated constriction pipettes (3, 10, or 30 nl) in a Mark I Liquid scintillation counter (Nuclear-Chicago Corp.) allowed for the measurement of the total amounts of radioactivity due to [14C]inulin and 42K injected. Isotope separation and overlap corrections between appropriately set counting windows were accomplished by the channel ratio method, applying appropriate corrections for *K decay. Assuming complete excretion of inulin, tubular efflux of 42K was calculated as the difference between injected and excreted moieties. Microinjection sites were selected for early distal localization by the administration of small amounts (0.04-0.06 ml) of a 5 % lissamine green solution (33), and care was taken to avoid retrograde flow during the injection of the isotope mixture. The magnitude of the injected sample was 3, 10, and 30 nl in high-K, low-sodium, and all other experimental conditions, respectively, and the mean duration of injections was 30 s (range 15-45 s). Urine was collected from the left ureter into counting vials containing 10 ml of Aquasol (New England Nuclear Corp., Boston, Mass.). Immediately following the injection, collections were made every 15 s for the first 2 min and every 30 s for an additional time period of 3 min. The mean inulin recovery of all injections was 96 %, and samples were not accepted if the total cumulative inulin recovery did not exceed 94 70. Data are expressed as mean values rf: standard error, and the Student t test was used to assess the statistical significance of differences. l
l
l
GIEBISCH,
AND
WHITTEMBURY
TABLE 1. Intratubular injections of [42K] and [‘4C]inulin---summary of renal excretion data _-____ .-~~-~-- ~ ~ -~ _-~---.~-~~~ _ ~_ .- -- __.-.--~~-_-.Exptl
Condition
l_____--l_. Control
I
High
K
High
K + Diamox
n
- --
Urine Flow, ml/min +kg __----~~
FE
- ----
Ns,
Na/In U/P _.-_-__
FEK, K/In U/P .---.-
---~
16
0.335~0.009
16
0.457&0.016
0.8837hO.0298
8
0.475jzO.016
1.510
jzO.0890
1.113
&0.0080
0.1075&0.0028
0.3220
~0 .0125
l
RESULTS
A summary of renal excretion data, including urine flow rates and fractional excretion rates of sodium and potassium, is represented in Tables 1, 2, and 3. Table 1 shows the results of experiments of those animals receiving a total tubular volume of injection of 30 nl. It is apparent that the
+
NCi2SO4
Low
K
KCI
12 32
0.375zt0.011
0.0800=t0.0013
highest urine flow rates were observed during stimulation of potassium excretion, achieved by the intravenous administration of an exogenous potassium load, by Diamox or sodium sulfate. As expected from the infusion of hypertonic sodium-containing solutions, the rate of fractional urinary sodium excretion is uniformly increased to levels between 8 and 15%. In contrast, fractional excretion rates of potassium vary and depend strongly on the experimental condition. Compared to control animals excreting an amount equivalent to some 32 %’ of the filtered potassium load, urinary potassium excretion is dramatically augmented in those groups of animals pretreated with a high-potassium diet and receiving potassium-containing solutions intravenously during the microinjection experiments. On the other hand, it is apparent that both a low-potassium diet or the administration of amiloride sharply reduced fractional urinary excretion rates to about one-fourth of the control values. Table 2 contains similar data on urine flow rates and fractional sodium and potassium excretion rates in control rats which, instead of 2.5 %I NaCl, received 2 %j mannitol at a rate of 1.6 ml . h-l -100 g-l. This infusion was chosen because in the rat it does not produce a marked natriuresis, and hence it allows for the demonstration of the effects of a low-sodium diet upon urinary sodium and potassium excretion rates. An injection volume of 10 nl was chosen, and the sodium concentration of the tubular injectate reduced to 10 meq/liter to avoid overloading of the tubular sodium transport mechanism. It has been demonstrated that dietary pretreatment with a low-sodium diet reduces both sodium and potassium excretion rates (11, 22). This was fully confirmed in the present study. Inspection of Table 2 indicates that in low-sodium animals the fractional sodium excretion rate drops to less than $&th of the control sodium excretion rate. Similarly, fractional potassium excretion rates are reduced to about one-sixth of control excretion rates. Table 3 summarizes data from a series of experiments similar to those summarized in Table 1, with the exception that in the control and high-K animals summarized in Table 3, the tubular rate of injection was lower by a factor of 10, i.e., 3 instead of 30 nl. It is apparent that fractional urinary excretion rates of potassium were greatly augmented above the level of excretion rate achieved under control conditions by the pretreatment with a high-K diet and the administration of the potassium-containing infusion fluids.
Downloaded from www.physiology.org/journal/ajplegacy at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
RENAL
TUBULAR
1229
K TRANSPORT
2. Intratubutar injections of [42K] and [W]z’nuhsummaryof renal excretion data
3Uni
TABLE
Exptl Condition
Urine Flow, ml/min * kg
s
ConrYoz 2
12
Low
20
Na
0.087&0.0058 035 *o. 0005
0
l
FEK,
FEN*,
&/In
60 -
U/P
K/In
0.02590~0.00150 0.00021*0.00002
U/P
0.3170~0.0013 0.0537&0.0028
VaIucs are means & SE. n, number of clearance periods. Animals received 2% mannitol at a rate of 1.6 ml/h. 100-g ralt. Injected volume, 10 nl. FEN,, fractional excretion ot wxlium; FEK, fractional excretion of potassium.
Conirol High
tL 3
K
u$e;1g.
FEN&,
Na/In
50 -
50 -
50
40 -
40 I-
40
30 -
30 -
30
z kz $
20 -
20 -
20
IO 1
IO -
IO
FEK, U/P
12
0.370~0.018
0.124&0.0047
0.250&0.0004
8
0.230&0.011
0 * 079 ho * 0003
0.650&0.0300
Vaiues are means & SE. n, number of clearance periods. NaCl at a rate of 3.2 ml/h* 100-g rat. Injected volume, 3 nl. cretion of sodium; FEK, fractional excretion of potassium.
100
R/In
U/P
Animals received FEN,, fractional
60 -
2I :: zz Y
TABLE 3. fntratubular injectionsof L4”K] and [14C]inulinsummaryof renal excretiondata
Exptl Condition
& Y
80
80
Y e
60
60
40
40
20
20
0
2.5% ex-
? s -I 2 z ae
Figures l-4 show representative examples of microinjection data obtained in the different control conditions and during various states of enhanced or reduced urinary potassium excretion rates. Table 4 summarizes mean recovery rates of 42K corrected for inulin loss (always less than 6 %) in the different experimental conditions. Figure 1 provides information on the excretion patterns of [%]inulin and 42K after early distal tubular injections in control animals. The top graph depicts fractional recovery rates, and the lower graphs depict total cumulative recoveries. Tubular microinjections were made at a rate of 3, 10, and 30 nl, respectively. It is apparent that excretion of [14C]inulin was complete within 3-4 min. Three points deserve mention. First, the excretion of both isotopes was somewhat delayed, although still reaching constant levels within 3-4 min in the control experiment in which an intravenous infusion of mannitol (2 % at 1.6 ml h-l - 100 g body wt-‘) was given instead of saline (2.5 %, NaCl at 3.2 ml 4h-l 100 g body wt-l). This was due no doubt to the lower urine flow rates of the animals receiving mannitol. (Compare mean urine flow rates in Tables 1, 2, and 3.) This indicates that reproducible excretion patterns obtain over the range of urine flow rates achieved in the present series of experiments. A second point concerns the total rate of 42K excretion rates in the three representative control experiments. It can be seen that the recovery of 42K is inversely proportional to the total volume, i.e., amount of l12K injected. Inspection of Table 4 shows that the mean ratio of excretion of 42K/[14C]inulin was 85.9, 74.9, and 62.0 % in the animals receiving 30-, lo-, and 3-nl injections. These differences are statistically significant (P < 0.01 : control experiments in which 30 and 10 nl were given; P < 0.005: control experiments in which 30 and 3 nl were given; P < 0.05 : control experiments in which 10 and 3 nl were given). Finally, the similarity of the inulin and potassium excretion patterns should be noticed. With respect to [lAC]inulin, the 42K recovery is only slightly delayed and in general, the excretion patterns of potassium follow closely that of inulin. A similar excretion pattern was also found by De Roufignac and Guinnebault (9). Thus, as defined by Gottschalk, l
80 60
I
40
20
t
0
0
I
0
MIN
l
3nI
IUnl
MIN
2
-.I
3
4
MIN
FIG. 1. Comparison of [14C]inulin and 4% excretion patterns after early distal tubular microinjections in rats on a control diet. TOP, Bottom. fractional recovery. Shaded area : 42K, open area: [14C]inulin. cumulative recovery. Open circles: [14C]inulin, filled circles: 42K reintravenous infusion: 2.5y0 NaCl at a rate of coveries. Left panel. injection volume: 30 nl. 3.2 ml-h- ‘400 g body wt- l. Intratubular Middle panel: intravenous infusion: 2% mannitol at 1.6 ml. h-l* 100 g body wt? Intratubular injection volume: 10 nl. Right panel: intravenous infusion: 2.5y0 NaCl at a rate of 3.2 ml h-l 100 gm body wt? Intratubular injection volume: 3 nl. l
l
Morel, and their associates (16, 28), potassium recovery is LCdirect,” i.e., with a time course similar to that of the simultaneously injected inulin. Figure 2 summarizes examples of the time course of [‘4C]inulin and 42K recovery in animals in which a total volume of 30 nl was injected into the early distal tubule and in which urinary potassium excretion rates diKered significantly from that in control rats. Compared to the control excretion patterns of inulin and potassium (see Fig. 1, left panel), both the time course and the extent of excretion of 42K was unchanged in animals in which potassium excretion was stimulated (high-K animals), whereas total recovery of injected 42K was significantly reduced, although not delayed, in animals deprived of dietary potassium lor several weeks (low-K animals) and in animals in which the rate of urinary potassium excretion had been curtailed by the prior administration of amiloride, a mildly diuretic agent known to inhibit potassium secretion at the distal tubular level (10). Inspection of Table 4 shows that the mean 42K/[14C]inulin ratios are similar in control I and high-K animals, whereas these ratios are significantly depressed in low-K and amiloride-treated animals (P < 0.001). Figure 3 compares 14C and 42K excretion patterns in animals receiving 2 % mannitol intravenously to stimulate urine flow rate. Sodium excretion rate was varied by dietary sodium withdrawal. On the left, a representative control excretion patterns is shown; on the right the recovery pattern of an experiment in which pretreatment with a lowsodium diet had led to significant reduction of both urinary sodium and potassium excretion. It is apparent that the 42K recovery is reduced in the low-sodium animals. The respec-
Downloaded from www.physiology.org/journal/ajplegacy at Glasgow Univ Lib (130.209.006.061) on February 13, 2019.
1230
FOWLER,
4. Recovery of microinjected [42K] compared to [‘qC]inulin excretion in diyerent experimental condz’tions -_I~-~
CONTROL
% 4$jjg$”
?z
I (30 nl)
85.93&2.42+
12
High
K (30 nl)
88.98kO.98
13
NW
High
K
82.18jz2.66
9
NSt
78.85A5.04
11
NW
55.96h5.40
14
