AMERICAN
JOURNAL
OF PHYSIOLOGY Printed
Vol. 229, No. 6, December 1975.
in U.S.A.
study of renal magnesium
Micropuncture
in magnesium-loaded
rats
MICHELE Assistance
G. BRUNETTE, N. VIGNEAULT, of M. Chan and F. Bertrand)
De/w-tment
of Pedia/rics,
Maisonneuve
Hospital,
AND
University
BRUNETTE, MICHELE G., N. VIGNEAULT, AND S. CARRIERE. Microfuncture study of renal magnesium transport in magnesium-loaded rats. Am. J. Physiol. 229(6) : 1695-1701. 1975.-Mg transport
in the deep loop of Henle was studied in 15 young rats (SO-SO g) after acute systemic Mg loading (UF,, (ultrafilterable Mg) 4.77 meq/liter). Intratubular Mg was measured with a recently described fluorometric microtechnique. The mean values of the TF/P inulin and TF/UF Mg ratios were 3.32 & 0.13 and 4.25 ZL 0.17, respectively. The proportion of filtered Mg recovered in this part of the nephron was therefore 131.2 =t 5.0 %, indicating that an appreciable amount of Mg entered the lumen prior to the sites of puncture. A significant correlation between the TF/P inulin and TF/UF Mg ratios suggests that water reabsorption also contributes to the high concentration of Mg in the loop of Henle during systemic Mg loading. In another series of young rats (90-160 g), similarly loaded with MgClz (UF,, 5.81 meq/ liter), Mg and inulin were measured in superficial proximal (PT) and distal tubules (DT). Punctures were paired at two sites of the same PT and DT. The Mg concentration increased progressively along the PT in such a way that 90.5y0 of the filtered load still remained in the late proximal loops. If superficial and deep proximal tubules behave in a similar manner, it may be concluded that the site of entry of Mg is located between the late accessible part of the PT and the bend of the loop of Henle. Only 58.0 * 3.0% of the filtered Mg was delivered to the DT, indicating that Mg is extensively reabsorbed in the ascending limb, despite systemic loading. The proportion of filtered Mg did not vary along the DT, indicating no net reabsorption. transport of electrolytes; distal reabsorption
loop of Henle;
transport
proximal
S. CARRIERE
of Montreal,
School
(With of Medicine,
the Technical Montreal,
Canada
comparing the Mg content of urine with the composition of distal tubular fluid after Mg loading in dogs and rats, Wen et al. (31) and Le Grimellec et al. (17) concluded that a net addition of Mg possibly occurs in the terrninal nephron segments. Here again, a comparison between superficial distal tubular fluid and final urine is not convincing. In hydropenic nonloaded rats, paired punctures performed at two different sites of the same distal tubule failed to detect any input of Mg into this part of the nephron (4) . If Mg enters into the tubular lumen at any level, i.e., the loop of Henle, the distal tubule, or the collecting duct, one would expect that systemic Mg loading enhances such mechanisms. Using a new microtechnique to measure Mg in tubular fluid (5), Mg transport was studied along the superficial proximal and distal tubules and the papillary loops of Henle in Mg-loaded rats. The results indicate that significant net entry of Mg occurs prior to the bend of the loop of Henle. This “secretion” is partially compensated by an intensive reabsorption in the ascending limb. Finally, no net movement of Mg was detected in the distal tubule.
reabsorption;
RECENT MICROPUNCTURE STUDIES performed in dogs (2, 31), rats (4, 16, 17, 20), and Psamommys (25) have provided considerable information concerning the tubular transport of magnesium (Mg). Some controversy, however, persists, particularly dealing with the physiology of this electrolyte in the loop of Henle. De Rouffignac (25) reported data suggesting a net addition of Mg to the descending limb in the Psamommys. Our laboratory (4) could not find any significant difference between the fraction of filtered Mg recovered ( %EhIg) in the late superficial proximal tubule and the loop of Henle of the deep nephrons. However, a comparison of superficial and deep nephron populations remains questionable and excludes any definite conclusion. The hypothesis of a Mg secretion in the distal part of the nephron also remains a subject of controversy. By
METHODS
SuperJicial proximal and distal tubule experiments. Experiments were performed in 22 fasted (16 h) male SpragueDawley rats, ranging from 90 to 160 g in weight. The animals were allowed free access to water prior to the study. After anesthesia with Inactin 80 mg/kg ip, a tracheostomy was performed, and the jugular vein and the carotid artery were cannulated for intravenous infusion, blood sampling, and arterial pressure recording. Following a priming dose of inulin (22 mg) and MgCl2 (450 peq) perfused within a period of 10 min, a sustaining infusion in half-Ringer lactate was started, at a rate of 0.025 ml/min, in order to achieve a plasma inulin concentration of 75100 mg/ 100 ml. The addition of MgCl2 (160-250 meq/liter) to this sustaining infusion maintained a plasma Mg value of approximately 6 meq/liter. Catheters were placed into the ureters, and the left kidney was prepared for micropuncture through a flank incision. Clearances of inulin and Mg were performed hourly. The fractional excretion of Mg was calculated using a value of ultrafiltrable over plasma Mg ratio of 80 % as previously determined in this laboratory using the anaerobic method of Toribara et al. (25). Recent microdosage of Mg
1695
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1696
BRUNETTE,
in the glomerular filtrate in Wistar rats of Munich has confirmed the validity of this ratio (5). In both the proximal and distal tubule experiments, punctures were paired, i.e., fluid from the same tubule was collected at two different sites, with maximum distance between these two sites. These loops were identified by a previous intratubular injection of lissamine green via a micropipette of 2 pm OD. The most distal puncture always preceded the earliest one in order to prevent leakage. All collections were performed with 4- to 5-pm-OD pipettes. Papilla experiments. Fifteen young rats (50-60 g) were used for micropuncture of the loop of Henle. The animals were anesthetized and perfused in the manner described for the previous series. The sustaining perfusion, however, was given at a slightly lower rate (0.019 ml/min). After a 60min perfusion period, a flank incision was made, and the left papilla was exposed according to the technique described by Jamison ( 10). The limbs of the loop of Henle were identified by their particular appearance on the surface of the papilla and confirmed by the injection of traces of lissamine green contained in the tip of the collecting pipette. The outside diameter of these pipettes ranged from 3 to 5 pm. Clearances of inulin and Mg were performed hourly on the control kidney. On the experimental side, urine was collected by capillarity through a fire-polished capillary tubing. These collections permitted the measurement of urine (U) inulin and UMs and a comparison of the urine-to-plasma inulin ratio ((U/P) rn), the urine-toultrafiltrable Mg ratio ((U/UF)Mp), and fractional excretion of Mg (FEM,) on both sides. Analytical methods. Plasma and urine Mg were analyzed by atomic absorption and inulin by the photometric method of Heyrowski (9). Tubular fluid inulin was measured using the fluorometric method of Vurek and Pegram (30). The intratubular Mg concentration was determined by a microfluorometric method recently set up in our laboratory (4, 5). This technique utilizes the fluorescent property of the complex Mg N, N’-bis-salicylidene-2 ,3-diaminobenzofuran and allows accurate measurement of Mg in 3-5 nl of tubular fluid. Simultaneous determination of Mg in 19 plasma ultrafiltrates by this micromethod and by atomic absorption gave a highly significant correlation (r = 0.97).
Clearance data. Table 1 presents the mean values obtained from the three types of experiments. Plasma ultrafiltrable Mg (UF& approximates 5 meq/liter, which corresponds 1. Clearance data
Type of Expts
UFaap, meqhter
Proximal n = 33
5.8 rto. 17
Loop of Henle n = 41
4.77 hO.11
Distal n = 43
5.49 =to. 17
AND CARRIERE
to a total plasma Mg varying from 6 to 7 meq/liter. We consider this level of Mg loading to be a maximum beyond which arterial pressure tends to decrease and mortality occurs. The GFR was equal for both kidneys in proximal and distal experiments. The lower value in the control kidney of papillary experiments (loop of Henle) may be explained by the younger age of the animals. In the same experiments, (U/P) In shows a tendency to decrease on the experimental side, when compared to the control side (83.3 =t 5.7 VS. 115.7 & 8.3). This well-known effect of the pyelic cavity opening has been explained recently by a lack of contact between urine and the papilla, thus preventing a normal backdiffusion of urea into the papilla (26). We have also observed that the circulation within the vasa recta slows down progressively after 1 h of papilla exposure, which may also contribute to the decreased concentrating capacity on this side. Mean fractional excretion of Mg remains between 5 1.5 and 62.4 %. On one occasion, a value over 100 % was observed ( 140 %). However, this high ratio occurred when the corresponding value of GFR was low. Since blood samples were taken only every hour, interpolation of plasma inulin values may explain this single event. The mean urine flow was approximately 7 pl/min in larger rats (cortical experiments) and 4 pl/min in smaller ones (papillary experiments). Indeed, in all experiments an abrupt increase of urine flow occurred when starting the Mg load. This increase probably does not correspond to volume expansion, since the hematocrit remained stable during the experiments, but rather is a result of the Mg loading. &perjicial proximal tubule. As observed with hydropenic nonloaded rats (4), Mg concentration increases along the length of the proximal tubule. Figure 1 represents the variations of the tubular fluid-to-ultrafiltrable Mg ratio ( (TF/UF)M,) with the tubular fluid-to-plasma inulin ratio are joined by straight lines ((TWP) In)- P aired punctures illustrating an evident increasing concentration with tubule distance. The regression line of all these data is highly significant (r = 0.37 + 0.75~) (P < 0.001). The calculated proportion of filtered Mg remaining at the site of puncture (%E,, (TF/UF)M, : (TF/P)rn) VS. (TF/P)In) is presented in Fig. 2. Here also, straight lines join paired punctures, but the scatter renders the interpretation difficult. However, values observed in the late part of the tubules are relatively more constant and approximate 90 %. Papillary loops of Henle. Forty-one punctures were performed in the loop of Henle, near the bend. In all but
RESULTS
TABLE
VIGNEAULT,
._ GFR, ml/min
W/UFhg
W/Phn
E
0.60 hO.03
0.66 hO.09
90.1 he.2
85.4 h3.4
51.9 ~2.5
50.7 zt2.4
62.0 zt4.3
60.2 h2.1
0.42 hO.02
83.3 zt5.7
115.7 h8.3
39.5 A2.4
61 .O *3.4
51.5 h2.5
56.7 h2.3
0.73 hO.02
111.9 zk3.9
99.8 h2.5
64.6 ~1.6
62.4 h1.3
59.4 zt1.9
62.4 bO.9
Values are means =t SE. E, experimental
E
Vol, jd/min
C
0.66 hO.02
C
F&g
E
C
E
C
E
C
6.8 hO.3
8.2 kO.4 3.9 AO.3
6.6 +O.l
7.4 AO.1
kidney; C,
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RENAL
MAGNESIUM
TRANSPORT
IN MAGNESIUM
LOADING
1697
7
6
5 2 4
5 2I-
3 TF/P
In.
1. Proximal tubule. Variations of (TF/UF)Q as a function data obtained by puncturing 2 sites of same in. Paired nephron (open circles) are joined by a straight line. Closed circles correspond to random unpaired punctures. FIG.
of
(‘WI3
2
1 1
I
I
I
I
I
2
3
4
5
6
TF/P FIG.
3. Loop
of
In.
Henle.
Correlation between (TF/UF)Q and is significant (P < 0.01). Most of values line of equality (not illustrated).
(TF/P) I~: this correlation are situated
0
L 1
I 2 TF/P
2. Proximal tubule. tubule (%EM~) are plotted data (open circles) are joined FIG.
1 3 In.
Fractions of filtered against corresponding by a straight line.
Mg remaining in (TF/P) I~. Paired
nine (Fig. 3, Table Z), the TF/UF ratio was greater than the corresponding (TF/P),,, indicating that Mg entered into the nephron prior to the site of puncture. Despite a certain degree of scatter, there is a good correlation between the two parameters, (TF/UF)M, and (TF/P) In (P < 0.01). In all the experiments, Mg was always more concentrated in urine than in the loop of Henle. Conversely, the proportion of filtered Mg remaining in the loop of Henle ( %E& was always much higher than the corresponding fractional excretion in urine (FEM,) (Table 2) . Superjicial distal tubule. In the distal as in the proximal tubule, Mg concentration increases with distance. Figure 4 represents the variations of (TF/UF),, as a function of Paired punctures are joined by a straight line. (TF/P) InThere is a general tendency for Mg to increase and the slope (y = 0.96 + 0.46~) is highly significant (P < 0.001). By contrast to the results obtained in hydropenic nonloaded rats, this slope seems constant along the distal tubule, and the Mg concentration ratio varies with (TF/P)r,. The fraction of filtered Mg which remains within the lumen ( %E& is therefore identical in the early and
above
late distal tubule and, interestingly, approximates the fractional excretion of Mg in urine (Fig. 5). A summary of the micropuncture results are presented in Table 3 and Fig. 6. In Table 3, proximal and distal tubules are arbitrarily separated into “early” and “late,” depending on the values of (TF/P) rn below or above 2 and 10, respectively. Whereas in the proximal tubule, (TF/UF)M B and (TF/P) In are closely related so that 90.5 =k 4.5 % of the filtered load is still present in the late segment, a net dissociation between the two parameters occurs in the 100p of Henle, where the mean ratio of (TF/UF)M, exceeds the mean (TF/P)rn (4.25 =t 0.17 vs. 3.32 & 0.13). The resulting %E,, is 13 1.2 & 5.0 %, a value significantly higher than the corresponding one in the late superficial proximal tubule (P < 0.001) and the early superficial distal tubule (P < 0.001). In the distal tubule, (TF/P),, and (TF/UF)M, increased from 8.71 & 1.31 and 4.72 & 0.074 to 18.17 & 1.54 and 9.3 1 & 0.9 1, respectively. Therefore, the resulting calculated %E, g remains at approximately 55 % along the distal tubule (58.0% & 3.0 in the early and 53.9% & 2.5 in the late segments). Figure 6 illustrates schematically the mean values of %EMg at the various sites of puncture in Mg-loaded rats and in hydropenic control animals. The latter data originate from recently published observations (4). The dashed lines draw attention to the fact that the data were obtained from different types of nephrons (deep and superficial). The %E,, in the glomerular filtrate is assumed to be 100 %, as recently demonstrated in Wistar rats of the Munich strain (5).
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1698
BRUNETTE, 2. Loop of Hen/e, detailed data - -__~ - ~_-_--_._--_-____~-- ~_-
TABLE
Rat No.
CJ.‘W’h
n
%EH,o
3.11
8
9
('WUFh,
32.2
-_____ FEarg,%
%hb,
3.33
107
E
C
57
66
3.46 5.80
28.9 17.2
4.48 5.27
129 90
60 38
68 44
3.66
27.3
5.13
140
54
63
4.62
21.7
3.69
80
68
61
3.32
30.1
3.35
101
23
39
2.40 2.32
41.7 43.1
2.52 3.0
105
53 43
48 42
2.53 2.14 2.57 2.39 2.53
39.5 46.7 38.9 41.8 39.5
3.93 3.91 4.76 3.25 3.89
182 185 135 153
69 67 65 63 60
83 74 70 67 62
3.31 3.30 3.92 5.04 4.14 5.62 4.83
30.2 30.3 25.5 24.2 17.8 20.7
3.46 4.76 4.73 6.98 4.37 4.60 4.48
104 144 120 138 105 81 92
22 22 23 23 23 24 24
31 31 33 34 36 39 39
3.58 3.34 2.97
27.9 29.9 33.7
4.03 4.27 4.16
112 127 140
51 52 53
47 49 50
19.8
VIGNEAULT,
129 155
Rat No.
W/WI,
%EH~o
AND CARRIERE
-_
CWUFh,
%Qg
Fhg,
%
E
C
2.85 3.73 3.0 2.63
35.1 26.8 33.3 38.0
7.16 5.07 4.16
196 191 169 158
54 55 53 55
51 52 50 52
II
3.67 3.51 2.55 2.90
27.3 28.5 39.2 34.5
6.15 6.16 4.30 4.73
167 175 168 163
59 61 61 62
72 72 72 72
I2
2.94 3.05
34.0 32.8
3.57 2.91
121 95
71 66
81 74
13
2.78
36.0
3.55
127
53
45
14
2.92 2.85 2.93
34.3 35.1 34.1
2.70 2.58 2.4Q
92 90 92
69 68 68
67 67 67
15
3.22 2.89 3.43 3.3
31.1 34.6 29.2 30.3
3.98 4.10 4.16 4.54
123 141 121 137
62 53 52 51
72 62 61 59
Mean SE SD
3.32 0.84 0.13 41
31.8 6.86 1.07 41
4.25 1.10 0.17 41
131 32 5 41
52 16 2.5 41
57 15 2.3 41
IO
n
5.59
TUBULAR FLUID
URINE X FE@
X EMg 90 80 70 -
g 0 Ii.‘I ..
60 50 40 30 20
1 1
0 L--
I 4
I
1
1
1
6
6
IO
12
,
I
I
I
I
1
14
16
16
20
22
24
TF/P
FIG. 4. Distal tubule. (TF/UF)M, Paired data (open circles), obtained distal tubule, are joined by a straight
In
are plotted by puncturing line.
against (TF/P)r,. 2 sites of same
30
28
26
24
22
20
18
16
14 12 10 X EHzO
8
6
4
2
2
1.0
0
FIG. 5. Distal tubule. Fractions of filtered Mg remaining in tubule (yOE~,) are plotted against corresponding (TF/P)rn. Paired data (open circles) are joined by a straight line. Corresponding urinary FEhlg are represented at right side of figure.
DISCUSSION
Transport of Mg in proximal tubule. Mg loading did not modify Mg transport in the proximal tubule. As observed with hydropenic nonloaded rats, Mg concentration increases progressively along this segment. However, the fraction of filtered Mg recovered in the late convolutions is higher (90.5 %) than in hydropenic rats (69 %), suggesting that only 10 % instead of 30 % of the filtered load has been reabsorbed. Since this load is approximately 4 times greater than in hydropenic rats, the absolute reabsorption did not change appreciably under the two experimental conditions. The rather low capacity of Mg transport in the rat proxi-
ma1 tubule in the presence of a positive electrical potential difference along most of its length (1, 7, 14) suggests a poor tubular permeability to this ion. Transport of Mg in loop of Henle. One hundred thirty percent of the filtered Mg is recovered in the bend of the loop of Henle. This finding proves that, in Mg-loaded rats, Mg enters into the tubular lumen somewhere prior to the bend of the loop of Henle. The finding of a value of %E,, greater than 100 % is original. Previous studies in nonloaded rats (4) have failed to demonstrate convincing evidence of Mg influx. Le Grimellec and co-workers (17) measured Mg in Mg-loaded rats, but conclusions concerning
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RENAL
MAGNESIUM
TRANSPORT
IN
3. Micropuncture data -.- . -__---_ __-__- ------_~--- _-.-___~__ --
TABLE
Site _.__
___-
n
- ___.
---
Early proximal (‘WPh,
Early distal < 10
(TF/P)r.
Late distal (TF/P)i, > 10 -_--.---.--Values are means
(‘WUFhrg -__
*o.
17
2.46 zto.07
41
%bxg
99.1 rt7.3
2.23 zto. 12
41.0 &l .o
90.5 zt4.5
3.32 Ito. 13
4.25 zto. 17
31.8 *l.l
131.2 3t5.0
18
6.87 zizo.50
3.92 ho.27
16.1 ztl.3
58.0 zt3.0
25
15.95 ztO.85 ---------
8.45 sto.50 - --- -.
6.7 zto.3
53.9 ~2.5
DEEP L.H.
1.46 10
%EH,o ~_-
68.4 zt2.2
-. -=t SE.
SUPERF I Cl AL PROXIMAL
n ---
1.49 kO.05
2
of Henle
LOADING
~-.- -- _. -
WV% -.--
MAGNESIUM
SUPERF I C I AL OISTAL
TOTAL URINE
90 80 2 w *
70 * 60 50 I I I
40 -
I I
30 20 10 0 Y g Loading Control
mm
68.4
41.0
31.8
16. i
6.7
0.88
68.5
43.1
23.6
19.2
8.9
0.44
%E Hz0
FIG. 6. Schematic illustration of mean y&, at various sites of puncture, in Mg-loaded rats, and in hydropenic nonloaded animals. Data concerning last series of experiments have been taken from Brunette et al. (4). Dashed lines join data obtained from 2 different types of nephron (superficial and deep).
the 100~ of Henle were derived from interpolation of proximal and distal data. z8Mg microinjection studies (3) also failed to show any direct passage of the isotope from the superficial capillary to the tubular lumen. These results may be explained in several ways. It is possible that the low specific activity of the 28Mg and therefore the high concentration of cold Mg ( 100 meq/liter) are responsible for the rapid diffusion, and dilution of both cold and radioactive Mg, resulting in the passage of unmeasurable tracer isotope into the tubular lumen. It is also possible that the microinjection technique evaluates an immediate passive backflux phenomenon, whereas a significant amount of Mg
1699 may enter the tubular lumen because of other slower mechanisms such as active transport, or by simple leakage from an intracellular pool. However, we think that the microinjection technique has not proven any direct shunt from the capillaries to the tubules because the injected 28Mg never reached that portion of the tubule that is permeable to Mg. The superficial capillaries are dispersed around superficial proximal and distal tubules, probably also around superficial loops of Henle, but do not reach the deep nephrons including the medullary loops. The question arises as to what segment of the deep nephron Mg enters. (We have no proof that such an entry exists in the superficial nephrons.) The site of Mg entry might theoretically be the proximal tubule. However, a reabsorption rather than a secretion was found in the superficial proximal tubule, and the hypothesis of a net Mg entry into the deep proximal tubule would suggest fundamental differences between the superficial and deep populations of nephron. The pars recta of the deep nephron could also be the site of cation input. Recent observations suggest that this segment behaves differently from the rest of the proximal tubule and possesses a negative electrical potential (11). Mg may also enter the tubular lumen in the descending limb of the loop of Henle. This hypothesis was recently advanced in studies performed in Psamommys. The authors demonstrated a better correlation in the loop of Henle between the total osmolarity and the concentration of most electrolytes than between osmolarity and inulin concentration ratio. They concluded that the hyperosmolarity was essentially due to recycling of electrolytes rather than to water removal. However, the lack of correlation between osmolarity and inulin in these experiments might be due to the variations of inulin concentration in the late proximal, from one nephron to the other as well as from one animal to the other. In contrast, the osmolarity of proximal tubular fluid remains constant. Consequently, water abstraction in the descending limb may result in a proportional concentration of all electrolytes, but the scatter of (TF/P)I, may hide any such correlation. Indeed, we found a significant correlation between (TF/UF)M, and (TF/P) in in the loop of Henle of our Mg-loaded animals, suggesting that water removal plays a partial role in concentrating electrolytes. Experiments using isolated tubule technique tend to demonstrate that water removal is the unique factor responsible for electrolyte concentration in the descending limb (12, 13, 22-24). Recently, Pennell and colleagues (2 1) reported that urea infusion into protein-depleted rats significantly increases water removal from the descending limb. However, water removal from the descending limb would not account for a ratio %EMg of 130 %, nor %E, of 113 %, as recently reported in hydropenic rats (15). Mg might diffuse from adjacent structures, such as the collecting duct or the ascending limb. Mg concentration is high in urine, but to date, no micropuncture study is available concerning Mg transport in this segment. On the contrary, there is evidence that the ascending limb is the major site of Mg reabsorption, in the Mg-loaded as well as in the hydropenic animal. The proportion of filtered Mg reabsorbed in the ascending limb cannot be evaluated precisely, since the data ob-
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1700 tained from the loop of Henle and the distal tubule correspond to the deep and superficial nephrons, respectively. However, we assume that the %E,, remaining in the bend of the superficial loops of Henle remains somewhere between 130 %, as found in the deep loops, and 90 %, as found at the end of the superficial proximal tubule. Since 58 % of the filtered Mg is recovered in the early distal convolution, 32-72 % of the filtered Mg is reabsorbed in the superficial ascending limb; and as the filtered load is 4 times that of the corresponding value in hydropenic rats, the absolute Mg transport in the ascending limb of Mg-loaded rats is several times greater than in hydropenic animals. Therefore, the ascending limb possesses a great capacity to adapt Mg transport to an increasing load of Mg and remains the major determinant of Mg urinary excretion. It is not known whether Mg loading modifies transtubular potential. If this one is positive as occurs in hydropenia (6, 23), this intensive transport of Mg could well be a passive phenomenon. Transport of Mg in distal tubule. Whereas a slight degree of Mg reabsorption seems to occur in the early segment of the distal tubule in hydropenic rats ‘(4), no such reabsorption was observed in Mg-loaded animals. The Mg concentration increases steadily along the distal tubule, proportional to in a constant ratio of the (TF/P)i, (Fig. 4), resulting %E,, in the whole segment. This ratio (58.0 & 3.0% in the early and 53.9 & 2.5 % in the late distal) is similar to
BRUNETTE,
VIGNEAULT,
AND
CARRIERE
the FEMg in urine (59.4 A 1.9) of the distal experiments. However, urine is a mixture of fluid arising from superficial and deep nephrons, and the validity of a comparison between superficial distal tubular fluid with total urine is debatable. These results suggest, but do not prove, an absence of Mg transport in the collecting duct. If systemic loading does not modify electrical potential in the distal tubule and the collecting duct, the negativity of tubular lumen (1, 8, 18, 19, 27, 28, 33) may be factor in explaining the lack of Mg reabsorption at these levels. In conclusion, systemic Mg loading resulted in a significant entry of Mg into the tubular lumen at a site prior to the bend of the loop of Henle of deep nephrons. This site is either the descending limb of the loop or the pars recta of the deep proximal tubule. The ascending limb of the loop of Henle retains a high capacity for Mg transport with systemic Mg loading. Finally, neither Mg reabsorption nor secretion has been detected in the distal tubule or in the collecting system. The authors are indebted to Dr. Jacques technical advice. This work was supported by the Medical Canada, Grant MA 4472, and by a grant from Foundation. Received
for publication
26 March
Diezi
for
his helpful
Research Council of the Canadian Kidney
1975.
REFERENCES 1. BARRATT, L. J., F. C. RECTOR, J. P. KOKKO, AND D. W. SELDIN. Variations of transepithelial potential difference along the rat proximal tubule. Ciin. Res. 2 I : 675, 1973. 2, BRUNETTE, M. G., S. F. WEN, AND J. H. DIRKS. Micropuncture study of magnesium reabsorption in the proximal tubule of the dog. Am. J. Physiol. 216: 1510-1516, 1969. 3. BRUNETTE, M. G., AND M. ARAS. A microinjection study of nephron permeability to calcium and magnesium. Am. J. Physiol. 221: 1442-1448, 1971. 4. BRUNETTE, M. G., N. VIGNEAULT, AND S. CARRIERE. Micropuncture study of magnesium transport along the nephron in the young rat. Am. J. Physiol. 227: 891-896, 1974. 5. BRUNETTE, M. G., AND M. E. CROCHET. A new fluorometric method for the determination of magnesium in renal tubular fluid. Anal. Biochem. 65 : 79-88, 1975. 6. BURG, M. B., AND N. GREEN. Function of the thick ascending limb of Henle’s loop. Am. J. Physioi. 224: 659-668, 1973. 7. CHIRITO, E., AND J. F. SEELY. Studies of the electrical potential difference in rat proximal tubule. Am. J. Physiol. 229: 72-80, 1975. 8. GIEBISCH, G., G. MALNIC, R. M. KLOSE, AND E. E. WINDHAGER. Effect of ionic substitutions on distal potential differences in the rat kidney. Am. J. Physiot. 211: 560-568, 1966. 9. HEYROWSKI, A. A new method for the determination of inulin in plasma and urine. Clin. Chim. Acta 1: 470474, 1956. 10. JAHISON, R. L. Micropuncture study of superficial and juxtamedullary nephrons in the rat. Am. J. Physiol. 218 : 46-55, 1970. 11. KAWAMURA, S., M. IMAI, AND J. P. Ko~o. Evidence for electrogenic transport processes in the two different types of proximal straight tubules. J. C&n. Invest. 53 : 39A, 1974. 12. KOKKO, J. P. Sodium chloride and water transport in the descending limb of Henle. J. C/in. Invest. 49 : 1839-l 846, 1970. 13. KOSCKO, J. P. Urea transport in the proximal tubule and the descending limb of Henle. J. C/in. Invest. 5 1: 1999-2008, 1972. 14. KOKKO, J. P. Proximal tubule potential difference: dependence on glucose HCOs, and amino acids. J. Clin. Invest. 52 : 13621367, 1973. 15. LACY, F. B., J. P. PENNELL, N. R. FREY, AND R. L. J AMISON. A comparison of potassium and sodium reabsorption in the descending limb of Henle’s loop in vivo. Clin. Res. 22 : 535A, 1974.
16. LE GRIMELLEC, C., N. ROINEL, AND F. MOREL. Simultaneous Mg, Ca, P, K, Na, Cl analysis in rat tubular fluid. 1. During perfusion of either inulin or ferrocyanide. Pjuegers Arch. 340: 181-196, 1973. 17. LE GRIMELLEC, C., N. ROINEL, AND F. MOREL. Simultaneous Mg, Ca, P, K, Na, and Cl analysis in rat tubular fluid. II. During Pjluegers Arch. 340 : 197-2 10, 1973. acute Mg plasma loading. 18. MALNIC, G., R. M. KLOSE, AND G. GIEBISCH. Micropuncture study of renal potassium excretion in the rat. Am. J. Physiol. 206: 674-686, 1964. Some electrical properties of 19. MALNIC, G., AND G. GIEBISCH. distal tubular epithelium in the rat. Am. J. Physiol. 223: 797808, 1972. 20. MOREL, F., N. ROINEL, AND C. LE GRIMELLEC. Electron probe analysis of tubular fluid composition. Nephron 6: 350-364, 1969. 21. PENNELL, J. P., V. SAJANA, N. R. FREY, AND R. L. J AMISON. The effect of urea infusion on the urinary concentrating mechanism in protein depleted rats. J. Clin. Invest. 55 : 399-409, 1975. 22. ROCHA, A. S., AND J. P. KOKKO. Transport of potassium in the proximal convoluted tubule and the descending limb of Henle. Clin. Res. 10: 607, 1972. 23. ROCHA, A. S., AND J. P. KOKKO. Sodium, chloride and water transport in the medullary thick ascending limb of Henle. J. Ciin. Invest. 52 : 612-623, 1973. 24. ROCHA, A. S., AND J. P. KOKKO. Membrane characteristics regulating potassium transport out of the isolated perfused descending limb of Henle. Kidney Intern. 4: 326-330, 1973. 25. ROUFFIONAC, C. DE., F. MOREL, N. Moss, AND N. ROINEL. Micropuncture study of water and electrolyte movements along the loop of Henle in Psamommys with special reference to magnesium, calcium and phosphorus. Pfruegers Arch. 344: 309-326, 1973. 26. SCHUTZ, W., AND J. SCHNERMANN. Pelvic urine composition as a determinant of inner medullary solute concentration and urine osmolarity. PJlucgers Arch. 334 : 154-l 66, 1972. 27. SOLOMON, S. Transtubular potential differences of rat kidney. J. Cellular Camp. Plrysiol. 49 : 351-365, 1957. 28. SULLIVAN, W. J. Electrical potential differences across distal renal tubules of Amflhiwna. Am. J. Physiol. 214: 1096-l 103, 1968. 29. TORIBARA, T. Y., P. TEREPKA, AND P. A. DEWEY. The ultra-
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 15, 2019.
RENAL filtrable normal
MAGNESIUM
TRANSPORT
IN
MAGNESIUM
calcium of human serum. I. Ultrafiltration values. J. Clin. Invest. 36 : 738-748, 1957. 30. VUREK, G. G., AND S. E. PEGRAM. Fluorometric determination of nanograms quantities of inulin. 16: 409419, 1966. 31. WEN, S. F., R. L. EVANSON, AND J. H. DIRKS.
1701
LOADING methods
method Anal.
and
for the Biochem.
Micropuncture
study of renal magnesium transport in proximal and distal of the dog. Am. J. Physiol. 219: 570-576, 1970. 32. WINDHAGER, E. E., AND G. GIEBISCH. Electrophysiology nephron. Physiol. Rev. 45 : 2 1+244, 1965. 33. WRIGHT, F. S. Increasing magnitude of electrical potential the renal distal tubule. Am. J. Physiol. 220 : 624-638, 197 1.
tubule of the along
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JOURNAL
OF PHYSIOLOGY Printed
Vol. 229, No. 6, December 1975.
in U.S.A.
study of renal magnesium
Micropuncture
in magnesium-loaded
rats
MICHELE Assistance
G. BRUNETTE, N. VIGNEAULT, of M. Chan and F. Bertrand)
De/w-tment
of Pedia/rics,
Maisonneuve
Hospital,
AND
University
BRUNETTE, MICHELE G., N. VIGNEAULT, AND S. CARRIERE. Microfuncture study of renal magnesium transport in magnesium-loaded rats. Am. J. Physiol. 229(6) : 1695-1701. 1975.-Mg transport
in the deep loop of Henle was studied in 15 young rats (SO-SO g) after acute systemic Mg loading (UF,, (ultrafilterable Mg) 4.77 meq/liter). Intratubular Mg was measured with a recently described fluorometric microtechnique. The mean values of the TF/P inulin and TF/UF Mg ratios were 3.32 & 0.13 and 4.25 ZL 0.17, respectively. The proportion of filtered Mg recovered in this part of the nephron was therefore 131.2 =t 5.0 %, indicating that an appreciable amount of Mg entered the lumen prior to the sites of puncture. A significant correlation between the TF/P inulin and TF/UF Mg ratios suggests that water reabsorption also contributes to the high concentration of Mg in the loop of Henle during systemic Mg loading. In another series of young rats (90-160 g), similarly loaded with MgClz (UF,, 5.81 meq/ liter), Mg and inulin were measured in superficial proximal (PT) and distal tubules (DT). Punctures were paired at two sites of the same PT and DT. The Mg concentration increased progressively along the PT in such a way that 90.5y0 of the filtered load still remained in the late proximal loops. If superficial and deep proximal tubules behave in a similar manner, it may be concluded that the site of entry of Mg is located between the late accessible part of the PT and the bend of the loop of Henle. Only 58.0 * 3.0% of the filtered Mg was delivered to the DT, indicating that Mg is extensively reabsorbed in the ascending limb, despite systemic loading. The proportion of filtered Mg did not vary along the DT, indicating no net reabsorption. transport of electrolytes; distal reabsorption
loop of Henle;
transport
proximal
S. CARRIERE
of Montreal,
School
(With of Medicine,
the Technical Montreal,
Canada
comparing the Mg content of urine with the composition of distal tubular fluid after Mg loading in dogs and rats, Wen et al. (31) and Le Grimellec et al. (17) concluded that a net addition of Mg possibly occurs in the terrninal nephron segments. Here again, a comparison between superficial distal tubular fluid and final urine is not convincing. In hydropenic nonloaded rats, paired punctures performed at two different sites of the same distal tubule failed to detect any input of Mg into this part of the nephron (4) . If Mg enters into the tubular lumen at any level, i.e., the loop of Henle, the distal tubule, or the collecting duct, one would expect that systemic Mg loading enhances such mechanisms. Using a new microtechnique to measure Mg in tubular fluid (5), Mg transport was studied along the superficial proximal and distal tubules and the papillary loops of Henle in Mg-loaded rats. The results indicate that significant net entry of Mg occurs prior to the bend of the loop of Henle. This “secretion” is partially compensated by an intensive reabsorption in the ascending limb. Finally, no net movement of Mg was detected in the distal tubule.
reabsorption;
RECENT MICROPUNCTURE STUDIES performed in dogs (2, 31), rats (4, 16, 17, 20), and Psamommys (25) have provided considerable information concerning the tubular transport of magnesium (Mg). Some controversy, however, persists, particularly dealing with the physiology of this electrolyte in the loop of Henle. De Rouffignac (25) reported data suggesting a net addition of Mg to the descending limb in the Psamommys. Our laboratory (4) could not find any significant difference between the fraction of filtered Mg recovered ( %EhIg) in the late superficial proximal tubule and the loop of Henle of the deep nephrons. However, a comparison of superficial and deep nephron populations remains questionable and excludes any definite conclusion. The hypothesis of a Mg secretion in the distal part of the nephron also remains a subject of controversy. By
METHODS
SuperJicial proximal and distal tubule experiments. Experiments were performed in 22 fasted (16 h) male SpragueDawley rats, ranging from 90 to 160 g in weight. The animals were allowed free access to water prior to the study. After anesthesia with Inactin 80 mg/kg ip, a tracheostomy was performed, and the jugular vein and the carotid artery were cannulated for intravenous infusion, blood sampling, and arterial pressure recording. Following a priming dose of inulin (22 mg) and MgCl2 (450 peq) perfused within a period of 10 min, a sustaining infusion in half-Ringer lactate was started, at a rate of 0.025 ml/min, in order to achieve a plasma inulin concentration of 75100 mg/ 100 ml. The addition of MgCl2 (160-250 meq/liter) to this sustaining infusion maintained a plasma Mg value of approximately 6 meq/liter. Catheters were placed into the ureters, and the left kidney was prepared for micropuncture through a flank incision. Clearances of inulin and Mg were performed hourly. The fractional excretion of Mg was calculated using a value of ultrafiltrable over plasma Mg ratio of 80 % as previously determined in this laboratory using the anaerobic method of Toribara et al. (25). Recent microdosage of Mg
1695
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1696
BRUNETTE,
in the glomerular filtrate in Wistar rats of Munich has confirmed the validity of this ratio (5). In both the proximal and distal tubule experiments, punctures were paired, i.e., fluid from the same tubule was collected at two different sites, with maximum distance between these two sites. These loops were identified by a previous intratubular injection of lissamine green via a micropipette of 2 pm OD. The most distal puncture always preceded the earliest one in order to prevent leakage. All collections were performed with 4- to 5-pm-OD pipettes. Papilla experiments. Fifteen young rats (50-60 g) were used for micropuncture of the loop of Henle. The animals were anesthetized and perfused in the manner described for the previous series. The sustaining perfusion, however, was given at a slightly lower rate (0.019 ml/min). After a 60min perfusion period, a flank incision was made, and the left papilla was exposed according to the technique described by Jamison ( 10). The limbs of the loop of Henle were identified by their particular appearance on the surface of the papilla and confirmed by the injection of traces of lissamine green contained in the tip of the collecting pipette. The outside diameter of these pipettes ranged from 3 to 5 pm. Clearances of inulin and Mg were performed hourly on the control kidney. On the experimental side, urine was collected by capillarity through a fire-polished capillary tubing. These collections permitted the measurement of urine (U) inulin and UMs and a comparison of the urine-to-plasma inulin ratio ((U/P) rn), the urine-toultrafiltrable Mg ratio ((U/UF)Mp), and fractional excretion of Mg (FEM,) on both sides. Analytical methods. Plasma and urine Mg were analyzed by atomic absorption and inulin by the photometric method of Heyrowski (9). Tubular fluid inulin was measured using the fluorometric method of Vurek and Pegram (30). The intratubular Mg concentration was determined by a microfluorometric method recently set up in our laboratory (4, 5). This technique utilizes the fluorescent property of the complex Mg N, N’-bis-salicylidene-2 ,3-diaminobenzofuran and allows accurate measurement of Mg in 3-5 nl of tubular fluid. Simultaneous determination of Mg in 19 plasma ultrafiltrates by this micromethod and by atomic absorption gave a highly significant correlation (r = 0.97).
Clearance data. Table 1 presents the mean values obtained from the three types of experiments. Plasma ultrafiltrable Mg (UF& approximates 5 meq/liter, which corresponds 1. Clearance data
Type of Expts
UFaap, meqhter
Proximal n = 33
5.8 rto. 17
Loop of Henle n = 41
4.77 hO.11
Distal n = 43
5.49 =to. 17
AND CARRIERE
to a total plasma Mg varying from 6 to 7 meq/liter. We consider this level of Mg loading to be a maximum beyond which arterial pressure tends to decrease and mortality occurs. The GFR was equal for both kidneys in proximal and distal experiments. The lower value in the control kidney of papillary experiments (loop of Henle) may be explained by the younger age of the animals. In the same experiments, (U/P) In shows a tendency to decrease on the experimental side, when compared to the control side (83.3 =t 5.7 VS. 115.7 & 8.3). This well-known effect of the pyelic cavity opening has been explained recently by a lack of contact between urine and the papilla, thus preventing a normal backdiffusion of urea into the papilla (26). We have also observed that the circulation within the vasa recta slows down progressively after 1 h of papilla exposure, which may also contribute to the decreased concentrating capacity on this side. Mean fractional excretion of Mg remains between 5 1.5 and 62.4 %. On one occasion, a value over 100 % was observed ( 140 %). However, this high ratio occurred when the corresponding value of GFR was low. Since blood samples were taken only every hour, interpolation of plasma inulin values may explain this single event. The mean urine flow was approximately 7 pl/min in larger rats (cortical experiments) and 4 pl/min in smaller ones (papillary experiments). Indeed, in all experiments an abrupt increase of urine flow occurred when starting the Mg load. This increase probably does not correspond to volume expansion, since the hematocrit remained stable during the experiments, but rather is a result of the Mg loading. &perjicial proximal tubule. As observed with hydropenic nonloaded rats (4), Mg concentration increases along the length of the proximal tubule. Figure 1 represents the variations of the tubular fluid-to-ultrafiltrable Mg ratio ( (TF/UF)M,) with the tubular fluid-to-plasma inulin ratio are joined by straight lines ((TWP) In)- P aired punctures illustrating an evident increasing concentration with tubule distance. The regression line of all these data is highly significant (r = 0.37 + 0.75~) (P < 0.001). The calculated proportion of filtered Mg remaining at the site of puncture (%E,, (TF/UF)M, : (TF/P)rn) VS. (TF/P)In) is presented in Fig. 2. Here also, straight lines join paired punctures, but the scatter renders the interpretation difficult. However, values observed in the late part of the tubules are relatively more constant and approximate 90 %. Papillary loops of Henle. Forty-one punctures were performed in the loop of Henle, near the bend. In all but
RESULTS
TABLE
VIGNEAULT,
._ GFR, ml/min
W/UFhg
W/Phn
E
0.60 hO.03
0.66 hO.09
90.1 he.2
85.4 h3.4
51.9 ~2.5
50.7 zt2.4
62.0 zt4.3
60.2 h2.1
0.42 hO.02
83.3 zt5.7
115.7 h8.3
39.5 A2.4
61 .O *3.4
51.5 h2.5
56.7 h2.3
0.73 hO.02
111.9 zk3.9
99.8 h2.5
64.6 ~1.6
62.4 h1.3
59.4 zt1.9
62.4 bO.9
Values are means =t SE. E, experimental
E
Vol, jd/min
C
0.66 hO.02
C
F&g
E
C
E
C
E
C
6.8 hO.3
8.2 kO.4 3.9 AO.3
6.6 +O.l
7.4 AO.1
kidney; C,
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RENAL
MAGNESIUM
TRANSPORT
IN MAGNESIUM
LOADING
1697
7
6
5 2 4
5 2I-
3 TF/P
In.
1. Proximal tubule. Variations of (TF/UF)Q as a function data obtained by puncturing 2 sites of same in. Paired nephron (open circles) are joined by a straight line. Closed circles correspond to random unpaired punctures. FIG.
of
(‘WI3
2
1 1
I
I
I
I
I
2
3
4
5
6
TF/P FIG.
3. Loop
of
In.
Henle.
Correlation between (TF/UF)Q and is significant (P < 0.01). Most of values line of equality (not illustrated).
(TF/P) I~: this correlation are situated
0
L 1
I 2 TF/P
2. Proximal tubule. tubule (%EM~) are plotted data (open circles) are joined FIG.
1 3 In.
Fractions of filtered against corresponding by a straight line.
Mg remaining in (TF/P) I~. Paired
nine (Fig. 3, Table Z), the TF/UF ratio was greater than the corresponding (TF/P),,, indicating that Mg entered into the nephron prior to the site of puncture. Despite a certain degree of scatter, there is a good correlation between the two parameters, (TF/UF)M, and (TF/P) In (P < 0.01). In all the experiments, Mg was always more concentrated in urine than in the loop of Henle. Conversely, the proportion of filtered Mg remaining in the loop of Henle ( %E& was always much higher than the corresponding fractional excretion in urine (FEM,) (Table 2) . Superjicial distal tubule. In the distal as in the proximal tubule, Mg concentration increases with distance. Figure 4 represents the variations of (TF/UF),, as a function of Paired punctures are joined by a straight line. (TF/P) InThere is a general tendency for Mg to increase and the slope (y = 0.96 + 0.46~) is highly significant (P < 0.001). By contrast to the results obtained in hydropenic nonloaded rats, this slope seems constant along the distal tubule, and the Mg concentration ratio varies with (TF/P)r,. The fraction of filtered Mg which remains within the lumen ( %E& is therefore identical in the early and
above
late distal tubule and, interestingly, approximates the fractional excretion of Mg in urine (Fig. 5). A summary of the micropuncture results are presented in Table 3 and Fig. 6. In Table 3, proximal and distal tubules are arbitrarily separated into “early” and “late,” depending on the values of (TF/P) rn below or above 2 and 10, respectively. Whereas in the proximal tubule, (TF/UF)M B and (TF/P) In are closely related so that 90.5 =k 4.5 % of the filtered load is still present in the late segment, a net dissociation between the two parameters occurs in the 100p of Henle, where the mean ratio of (TF/UF)M, exceeds the mean (TF/P)rn (4.25 =t 0.17 vs. 3.32 & 0.13). The resulting %E,, is 13 1.2 & 5.0 %, a value significantly higher than the corresponding one in the late superficial proximal tubule (P < 0.001) and the early superficial distal tubule (P < 0.001). In the distal tubule, (TF/P),, and (TF/UF)M, increased from 8.71 & 1.31 and 4.72 & 0.074 to 18.17 & 1.54 and 9.3 1 & 0.9 1, respectively. Therefore, the resulting calculated %E, g remains at approximately 55 % along the distal tubule (58.0% & 3.0 in the early and 53.9% & 2.5 in the late segments). Figure 6 illustrates schematically the mean values of %EMg at the various sites of puncture in Mg-loaded rats and in hydropenic control animals. The latter data originate from recently published observations (4). The dashed lines draw attention to the fact that the data were obtained from different types of nephrons (deep and superficial). The %E,, in the glomerular filtrate is assumed to be 100 %, as recently demonstrated in Wistar rats of the Munich strain (5).
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1698
BRUNETTE, 2. Loop of Hen/e, detailed data - -__~ - ~_-_--_._--_-____~-- ~_-
TABLE
Rat No.
CJ.‘W’h
n
%EH,o
3.11
8
9
('WUFh,
32.2
-_____ FEarg,%
%hb,
3.33
107
E
C
57
66
3.46 5.80
28.9 17.2
4.48 5.27
129 90
60 38
68 44
3.66
27.3
5.13
140
54
63
4.62
21.7
3.69
80
68
61
3.32
30.1
3.35
101
23
39
2.40 2.32
41.7 43.1
2.52 3.0
105
53 43
48 42
2.53 2.14 2.57 2.39 2.53
39.5 46.7 38.9 41.8 39.5
3.93 3.91 4.76 3.25 3.89
182 185 135 153
69 67 65 63 60
83 74 70 67 62
3.31 3.30 3.92 5.04 4.14 5.62 4.83
30.2 30.3 25.5 24.2 17.8 20.7
3.46 4.76 4.73 6.98 4.37 4.60 4.48
104 144 120 138 105 81 92
22 22 23 23 23 24 24
31 31 33 34 36 39 39
3.58 3.34 2.97
27.9 29.9 33.7
4.03 4.27 4.16
112 127 140
51 52 53
47 49 50
19.8
VIGNEAULT,
129 155
Rat No.
W/WI,
%EH~o
AND CARRIERE
-_
CWUFh,
%Qg
Fhg,
%
E
C
2.85 3.73 3.0 2.63
35.1 26.8 33.3 38.0
7.16 5.07 4.16
196 191 169 158
54 55 53 55
51 52 50 52
II
3.67 3.51 2.55 2.90
27.3 28.5 39.2 34.5
6.15 6.16 4.30 4.73
167 175 168 163
59 61 61 62
72 72 72 72
I2
2.94 3.05
34.0 32.8
3.57 2.91
121 95
71 66
81 74
13
2.78
36.0
3.55
127
53
45
14
2.92 2.85 2.93
34.3 35.1 34.1
2.70 2.58 2.4Q
92 90 92
69 68 68
67 67 67
15
3.22 2.89 3.43 3.3
31.1 34.6 29.2 30.3
3.98 4.10 4.16 4.54
123 141 121 137
62 53 52 51
72 62 61 59
Mean SE SD
3.32 0.84 0.13 41
31.8 6.86 1.07 41
4.25 1.10 0.17 41
131 32 5 41
52 16 2.5 41
57 15 2.3 41
IO
n
5.59
TUBULAR FLUID
URINE X FE@
X EMg 90 80 70 -
g 0 Ii.‘I ..
60 50 40 30 20
1 1
0 L--
I 4
I
1
1
1
6
6
IO
12
,
I
I
I
I
1
14
16
16
20
22
24
TF/P
FIG. 4. Distal tubule. (TF/UF)M, Paired data (open circles), obtained distal tubule, are joined by a straight
In
are plotted by puncturing line.
against (TF/P)r,. 2 sites of same
30
28
26
24
22
20
18
16
14 12 10 X EHzO
8
6
4
2
2
1.0
0
FIG. 5. Distal tubule. Fractions of filtered Mg remaining in tubule (yOE~,) are plotted against corresponding (TF/P)rn. Paired data (open circles) are joined by a straight line. Corresponding urinary FEhlg are represented at right side of figure.
DISCUSSION
Transport of Mg in proximal tubule. Mg loading did not modify Mg transport in the proximal tubule. As observed with hydropenic nonloaded rats, Mg concentration increases progressively along this segment. However, the fraction of filtered Mg recovered in the late convolutions is higher (90.5 %) than in hydropenic rats (69 %), suggesting that only 10 % instead of 30 % of the filtered load has been reabsorbed. Since this load is approximately 4 times greater than in hydropenic rats, the absolute reabsorption did not change appreciably under the two experimental conditions. The rather low capacity of Mg transport in the rat proxi-
ma1 tubule in the presence of a positive electrical potential difference along most of its length (1, 7, 14) suggests a poor tubular permeability to this ion. Transport of Mg in loop of Henle. One hundred thirty percent of the filtered Mg is recovered in the bend of the loop of Henle. This finding proves that, in Mg-loaded rats, Mg enters into the tubular lumen somewhere prior to the bend of the loop of Henle. The finding of a value of %E,, greater than 100 % is original. Previous studies in nonloaded rats (4) have failed to demonstrate convincing evidence of Mg influx. Le Grimellec and co-workers (17) measured Mg in Mg-loaded rats, but conclusions concerning
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RENAL
MAGNESIUM
TRANSPORT
IN
3. Micropuncture data -.- . -__---_ __-__- ------_~--- _-.-___~__ --
TABLE
Site _.__
___-
n
- ___.
---
Early proximal (‘WPh,
Early distal < 10
(TF/P)r.
Late distal (TF/P)i, > 10 -_--.---.--Values are means
(‘WUFhrg -__
*o.
17
2.46 zto.07
41
%bxg
99.1 rt7.3
2.23 zto. 12
41.0 &l .o
90.5 zt4.5
3.32 Ito. 13
4.25 zto. 17
31.8 *l.l
131.2 3t5.0
18
6.87 zizo.50
3.92 ho.27
16.1 ztl.3
58.0 zt3.0
25
15.95 ztO.85 ---------
8.45 sto.50 - --- -.
6.7 zto.3
53.9 ~2.5
DEEP L.H.
1.46 10
%EH,o ~_-
68.4 zt2.2
-. -=t SE.
SUPERF I Cl AL PROXIMAL
n ---
1.49 kO.05
2
of Henle
LOADING
~-.- -- _. -
WV% -.--
MAGNESIUM
SUPERF I C I AL OISTAL
TOTAL URINE
90 80 2 w *
70 * 60 50 I I I
40 -
I I
30 20 10 0 Y g Loading Control
mm
68.4
41.0
31.8
16. i
6.7
0.88
68.5
43.1
23.6
19.2
8.9
0.44
%E Hz0
FIG. 6. Schematic illustration of mean y&, at various sites of puncture, in Mg-loaded rats, and in hydropenic nonloaded animals. Data concerning last series of experiments have been taken from Brunette et al. (4). Dashed lines join data obtained from 2 different types of nephron (superficial and deep).
the 100~ of Henle were derived from interpolation of proximal and distal data. z8Mg microinjection studies (3) also failed to show any direct passage of the isotope from the superficial capillary to the tubular lumen. These results may be explained in several ways. It is possible that the low specific activity of the 28Mg and therefore the high concentration of cold Mg ( 100 meq/liter) are responsible for the rapid diffusion, and dilution of both cold and radioactive Mg, resulting in the passage of unmeasurable tracer isotope into the tubular lumen. It is also possible that the microinjection technique evaluates an immediate passive backflux phenomenon, whereas a significant amount of Mg
1699 may enter the tubular lumen because of other slower mechanisms such as active transport, or by simple leakage from an intracellular pool. However, we think that the microinjection technique has not proven any direct shunt from the capillaries to the tubules because the injected 28Mg never reached that portion of the tubule that is permeable to Mg. The superficial capillaries are dispersed around superficial proximal and distal tubules, probably also around superficial loops of Henle, but do not reach the deep nephrons including the medullary loops. The question arises as to what segment of the deep nephron Mg enters. (We have no proof that such an entry exists in the superficial nephrons.) The site of Mg entry might theoretically be the proximal tubule. However, a reabsorption rather than a secretion was found in the superficial proximal tubule, and the hypothesis of a net Mg entry into the deep proximal tubule would suggest fundamental differences between the superficial and deep populations of nephron. The pars recta of the deep nephron could also be the site of cation input. Recent observations suggest that this segment behaves differently from the rest of the proximal tubule and possesses a negative electrical potential (11). Mg may also enter the tubular lumen in the descending limb of the loop of Henle. This hypothesis was recently advanced in studies performed in Psamommys. The authors demonstrated a better correlation in the loop of Henle between the total osmolarity and the concentration of most electrolytes than between osmolarity and inulin concentration ratio. They concluded that the hyperosmolarity was essentially due to recycling of electrolytes rather than to water removal. However, the lack of correlation between osmolarity and inulin in these experiments might be due to the variations of inulin concentration in the late proximal, from one nephron to the other as well as from one animal to the other. In contrast, the osmolarity of proximal tubular fluid remains constant. Consequently, water abstraction in the descending limb may result in a proportional concentration of all electrolytes, but the scatter of (TF/P)I, may hide any such correlation. Indeed, we found a significant correlation between (TF/UF)M, and (TF/P) in in the loop of Henle of our Mg-loaded animals, suggesting that water removal plays a partial role in concentrating electrolytes. Experiments using isolated tubule technique tend to demonstrate that water removal is the unique factor responsible for electrolyte concentration in the descending limb (12, 13, 22-24). Recently, Pennell and colleagues (2 1) reported that urea infusion into protein-depleted rats significantly increases water removal from the descending limb. However, water removal from the descending limb would not account for a ratio %EMg of 130 %, nor %E, of 113 %, as recently reported in hydropenic rats (15). Mg might diffuse from adjacent structures, such as the collecting duct or the ascending limb. Mg concentration is high in urine, but to date, no micropuncture study is available concerning Mg transport in this segment. On the contrary, there is evidence that the ascending limb is the major site of Mg reabsorption, in the Mg-loaded as well as in the hydropenic animal. The proportion of filtered Mg reabsorbed in the ascending limb cannot be evaluated precisely, since the data ob-
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1700 tained from the loop of Henle and the distal tubule correspond to the deep and superficial nephrons, respectively. However, we assume that the %E,, remaining in the bend of the superficial loops of Henle remains somewhere between 130 %, as found in the deep loops, and 90 %, as found at the end of the superficial proximal tubule. Since 58 % of the filtered Mg is recovered in the early distal convolution, 32-72 % of the filtered Mg is reabsorbed in the superficial ascending limb; and as the filtered load is 4 times that of the corresponding value in hydropenic rats, the absolute Mg transport in the ascending limb of Mg-loaded rats is several times greater than in hydropenic animals. Therefore, the ascending limb possesses a great capacity to adapt Mg transport to an increasing load of Mg and remains the major determinant of Mg urinary excretion. It is not known whether Mg loading modifies transtubular potential. If this one is positive as occurs in hydropenia (6, 23), this intensive transport of Mg could well be a passive phenomenon. Transport of Mg in distal tubule. Whereas a slight degree of Mg reabsorption seems to occur in the early segment of the distal tubule in hydropenic rats ‘(4), no such reabsorption was observed in Mg-loaded animals. The Mg concentration increases steadily along the distal tubule, proportional to in a constant ratio of the (TF/P)i, (Fig. 4), resulting %E,, in the whole segment. This ratio (58.0 & 3.0% in the early and 53.9 & 2.5 % in the late distal) is similar to
BRUNETTE,
VIGNEAULT,
AND
CARRIERE
the FEMg in urine (59.4 A 1.9) of the distal experiments. However, urine is a mixture of fluid arising from superficial and deep nephrons, and the validity of a comparison between superficial distal tubular fluid with total urine is debatable. These results suggest, but do not prove, an absence of Mg transport in the collecting duct. If systemic loading does not modify electrical potential in the distal tubule and the collecting duct, the negativity of tubular lumen (1, 8, 18, 19, 27, 28, 33) may be factor in explaining the lack of Mg reabsorption at these levels. In conclusion, systemic Mg loading resulted in a significant entry of Mg into the tubular lumen at a site prior to the bend of the loop of Henle of deep nephrons. This site is either the descending limb of the loop or the pars recta of the deep proximal tubule. The ascending limb of the loop of Henle retains a high capacity for Mg transport with systemic Mg loading. Finally, neither Mg reabsorption nor secretion has been detected in the distal tubule or in the collecting system. The authors are indebted to Dr. Jacques technical advice. This work was supported by the Medical Canada, Grant MA 4472, and by a grant from Foundation. Received
for publication
26 March
Diezi
for
his helpful
Research Council of the Canadian Kidney
1975.
REFERENCES 1. BARRATT, L. J., F. C. RECTOR, J. P. KOKKO, AND D. W. SELDIN. Variations of transepithelial potential difference along the rat proximal tubule. Ciin. Res. 2 I : 675, 1973. 2, BRUNETTE, M. G., S. F. WEN, AND J. H. DIRKS. Micropuncture study of magnesium reabsorption in the proximal tubule of the dog. Am. J. Physiol. 216: 1510-1516, 1969. 3. BRUNETTE, M. G., AND M. ARAS. A microinjection study of nephron permeability to calcium and magnesium. Am. J. Physiol. 221: 1442-1448, 1971. 4. BRUNETTE, M. G., N. VIGNEAULT, AND S. CARRIERE. Micropuncture study of magnesium transport along the nephron in the young rat. Am. J. Physiol. 227: 891-896, 1974. 5. BRUNETTE, M. G., AND M. E. CROCHET. A new fluorometric method for the determination of magnesium in renal tubular fluid. Anal. Biochem. 65 : 79-88, 1975. 6. BURG, M. B., AND N. GREEN. Function of the thick ascending limb of Henle’s loop. Am. J. Physioi. 224: 659-668, 1973. 7. CHIRITO, E., AND J. F. SEELY. Studies of the electrical potential difference in rat proximal tubule. Am. J. Physiol. 229: 72-80, 1975. 8. GIEBISCH, G., G. MALNIC, R. M. KLOSE, AND E. E. WINDHAGER. Effect of ionic substitutions on distal potential differences in the rat kidney. Am. J. Physiot. 211: 560-568, 1966. 9. HEYROWSKI, A. A new method for the determination of inulin in plasma and urine. Clin. Chim. Acta 1: 470474, 1956. 10. JAHISON, R. L. Micropuncture study of superficial and juxtamedullary nephrons in the rat. Am. J. Physiol. 218 : 46-55, 1970. 11. KAWAMURA, S., M. IMAI, AND J. P. Ko~o. Evidence for electrogenic transport processes in the two different types of proximal straight tubules. J. C&n. Invest. 53 : 39A, 1974. 12. KOKKO, J. P. Sodium chloride and water transport in the descending limb of Henle. J. C/in. Invest. 49 : 1839-l 846, 1970. 13. KOSCKO, J. P. Urea transport in the proximal tubule and the descending limb of Henle. J. C/in. Invest. 5 1: 1999-2008, 1972. 14. KOKKO, J. P. Proximal tubule potential difference: dependence on glucose HCOs, and amino acids. J. Clin. Invest. 52 : 13621367, 1973. 15. LACY, F. B., J. P. PENNELL, N. R. FREY, AND R. L. J AMISON. A comparison of potassium and sodium reabsorption in the descending limb of Henle’s loop in vivo. Clin. Res. 22 : 535A, 1974.
16. LE GRIMELLEC, C., N. ROINEL, AND F. MOREL. Simultaneous Mg, Ca, P, K, Na, Cl analysis in rat tubular fluid. 1. During perfusion of either inulin or ferrocyanide. Pjuegers Arch. 340: 181-196, 1973. 17. LE GRIMELLEC, C., N. ROINEL, AND F. MOREL. Simultaneous Mg, Ca, P, K, Na, and Cl analysis in rat tubular fluid. II. During Pjluegers Arch. 340 : 197-2 10, 1973. acute Mg plasma loading. 18. MALNIC, G., R. M. KLOSE, AND G. GIEBISCH. Micropuncture study of renal potassium excretion in the rat. Am. J. Physiol. 206: 674-686, 1964. Some electrical properties of 19. MALNIC, G., AND G. GIEBISCH. distal tubular epithelium in the rat. Am. J. Physiol. 223: 797808, 1972. 20. MOREL, F., N. ROINEL, AND C. LE GRIMELLEC. Electron probe analysis of tubular fluid composition. Nephron 6: 350-364, 1969. 21. PENNELL, J. P., V. SAJANA, N. R. FREY, AND R. L. J AMISON. The effect of urea infusion on the urinary concentrating mechanism in protein depleted rats. J. Clin. Invest. 55 : 399-409, 1975. 22. ROCHA, A. S., AND J. P. KOKKO. Transport of potassium in the proximal convoluted tubule and the descending limb of Henle. Clin. Res. 10: 607, 1972. 23. ROCHA, A. S., AND J. P. KOKKO. Sodium, chloride and water transport in the medullary thick ascending limb of Henle. J. Ciin. Invest. 52 : 612-623, 1973. 24. ROCHA, A. S., AND J. P. KOKKO. Membrane characteristics regulating potassium transport out of the isolated perfused descending limb of Henle. Kidney Intern. 4: 326-330, 1973. 25. ROUFFIONAC, C. DE., F. MOREL, N. Moss, AND N. ROINEL. Micropuncture study of water and electrolyte movements along the loop of Henle in Psamommys with special reference to magnesium, calcium and phosphorus. Pfruegers Arch. 344: 309-326, 1973. 26. SCHUTZ, W., AND J. SCHNERMANN. Pelvic urine composition as a determinant of inner medullary solute concentration and urine osmolarity. PJlucgers Arch. 334 : 154-l 66, 1972. 27. SOLOMON, S. Transtubular potential differences of rat kidney. J. Cellular Camp. Plrysiol. 49 : 351-365, 1957. 28. SULLIVAN, W. J. Electrical potential differences across distal renal tubules of Amflhiwna. Am. J. Physiol. 214: 1096-l 103, 1968. 29. TORIBARA, T. Y., P. TEREPKA, AND P. A. DEWEY. The ultra-
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 15, 2019.
RENAL filtrable normal
MAGNESIUM
TRANSPORT
IN
MAGNESIUM
calcium of human serum. I. Ultrafiltration values. J. Clin. Invest. 36 : 738-748, 1957. 30. VUREK, G. G., AND S. E. PEGRAM. Fluorometric determination of nanograms quantities of inulin. 16: 409419, 1966. 31. WEN, S. F., R. L. EVANSON, AND J. H. DIRKS.
1701
LOADING methods
method Anal.
and
for the Biochem.
Micropuncture
study of renal magnesium transport in proximal and distal of the dog. Am. J. Physiol. 219: 570-576, 1970. 32. WINDHAGER, E. E., AND G. GIEBISCH. Electrophysiology nephron. Physiol. Rev. 45 : 2 1+244, 1965. 33. WRIGHT, F. S. Increasing magnitude of electrical potential the renal distal tubule. Am. J. Physiol. 220 : 624-638, 197 1.
tubule of the along
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