Phosphatase activity along the nephron of mice with hypophosphatemic vitamin-D-resistant rickets

Kidney International, Vol. 20 (1981) pp. 181—187

Phosphatase activity along the nephron of mice with hypophosphatemic vitamin-D-resistant rickets MICHELE G. BRUNETTE, MEANTHAN CHAN, and MANON LEBRUN Department of Pediatrics, Maisonneuve-Rose,nont Hospital, School of Medicine. University of Montreal, Montreal, Canada

Phosphatase activity along the nephron of mice with hypophosphatemic vitamin-D-resistant rickets. It has recently been suggested that alkaline

phosphatase may be involved in the renal transport of inorganic phosphorus (Pi). Because a specific defect of Pi reabsorption in the proximal tubule is detected in genetic hypophosphatemic vitamin-Dresistant rachitic mice, we measured the alkaline phosphatase activity along the nephron of this mutant strain, using an in vitro microassay of

alkaline phosphatase on single tubules. In a series of preliminary experiments performed in normal mice, enzyme activity was detected in glomeruli (1.28 [5EM] 0.08 pmoles of hydrolyzed substrate per millimeter of glomerulus per 30 mm of incubation), in the initial part of 1.04), and to a the proximal tubule (S1) (in pmoles/mm/30 mm: 20.3 lesser extent in the middle part of this same segment (S2) (13.8 1.1). No enzyme activity was detected in the late pars recta (S3) or in the loop of Henle, the distal tubule, and the collecting tubule. In 24 experiments performed in rachitic mice paired with normal mice, alkaline phosphatase activity was 14.8 0.8 vs 19.7 1.2 inS1; 13.3 1.3 vs. 13.8 1.0 in S2, and 0.1 0.5 vs. 0.7 0.5 in S3. in the two series of animals, respectively. Thus, in rachitic mice, the enzyme activity is significantly lower in S3 compared with the corresponding normal value (P < 0.005). This difference is not due to variations in nephron diameters because it remains significant when enzyme activities are expressed as picomoles

of dephosphorylated substrate per microgram of tubular protein (P < 0.02). It is not due to variations in ages between the animals because phosphatase activity per microgram of tubular protein does not significantly vary within the age range of our mice. Because of the renal leak of Pi in the rachitic mice, these animals are chronically Pi depleted. To compare the alkaline phosphatase activity in hypophosphatemic mice with the activity which should normally result from a comparable Pi depletion, that enzyme was measured in a separate series of 14 rachitic mice paired with genetically normal animals depleted of Pi for 7 to 14 1.2 vs. 14.4 0.9 days. Alkaline phosphatase activity in S was 24.6 in Pi-depleted and rachitic mice, respectively (P < 0.001). Pi depletion in normal mice increased rather than decreased the enzyme activity in

S, therefore accentuating the abnormality observed in the rachitic mice.

L'activité de phosphatase alcaline le long du nephron de souris chez rachitisme vitamino-sistant avec hypophosphatémie. Plusieurs travaux ont récemment suggéré que Ia phosphatase alcalinejoue un role dans Ic transport tubulaire renal du phosphore inorganique. Par ailleurs. un défaut specifique de cc transport fut mis en evidence au niveau du tube

1.3 vs. 13.8 1.0 dans S2, et 0.1 0.5 vs. 0.7 0.5 dans S, chez les deux series d'animaux, respectivement. Chez Ia souris rachitique.

l'activité enzymatique est done significativement abaissée au niveau de S3 en comparaison de Ia valeur correspondante chez l'animal normal (P <0.005). Cette difference n'est pas due a des variations de diamètre des néphrons puisqu'elle persiste lorsque les activités enzymatiques sont

exprimées en pmoles de substrat dephosphoryle par g de protéines tubulaires (P < 0.02). Elle n'est pas due non plus a des variations d'hge

des animaux puisque l'activité phosphatasique par i.g de protéines tubulaires ne vane pas de facon significative en dedans des limites d'hge de nos souris. A cause de Ia fuite rénale de phosphore que présentent

les sounis rachitiques, ces animaux en sont chroniquement depletes. Afin de comparer l'activité phosphatasique des sounis rachitiques avec celle que devrait entrainer normalement une depICtion en phosphore analogue, l'enzyme fut dosée chez une nouvelle sénie de 14 souris rachitiques pairées avec des souris génétiquement normales deplétées en phosphore durant 7 a l4jours. L'activité de Ia phosphatase alcaline au niveau de S fut de 24.6 1.2 vs. 14.4 0.9 chez les sounis normales déplétées en phosphore et chez les souris rachitiques respectivement (P < 0.01). La depICtion en phosphore augmente plutOt qu'elle ne baisse l'activité phosphatasique en S, ce qui accentue l'anomalie observée chez Ia souris rachitique.

Since the discovery of a mutant strain of mice presenting a syndrome of vitamin-D-resistant hypophosphatemic rickets by Eicher et al [1], information has accumulated concerning the pathogenesis of this disease. The relatively high phosphaturia that characterizes the disease in mice, as well as in man, results from a defect in the transport of inorganic phosphorus in the proximal convoluted tubule [2] and more precisely across the brush border membrane [3, 41. The defect involves sodiumdependent phosphorus transport. But, the precise molecular defect remains unknown. In addition, it is not evident whether

the transport defect across the brush border constitutes the primary disorder or is secondary to an abnormality situated

proximal de Ia souche mutante de souris présentant un rachitisme vitamino-résistant avec hypophosphatemie. Nous avons done mesuré l'activité phosphatasique Ic long du néphron de cette souche mutante, a l'aide d'une microtechnique enzymatique in vitro sur tubule isolé. Dans une série d'experiences préliminaires chez des souris normales, une activité enzymatique fut trouvée dans les glomerules: 1.28 (5EM) 0.08 pmoles de substrat hydrolyse par glomerule par 30 mm d'incubation; dans Ia partie initiale du tube proximal ou S (pmoles/mm/30 mm): 20.3 1.04; eta un degre moindre, dans la partie moyenne de ce segment ou 1.1. Aucune activité enzymatique ne fut detectée dans Ia S2: 13.8 portion terminale de Ia pars recta ou S, ni dans I'anse de Henle, le tube

distal ou le tube collecteur. Dans 24 experiences effectuées chez des souris rachitiques pairées avec des souris normales l'activité de Ia phosphatase alcaline était de 14.8 0.8 vs. 19.7 1.2 dans S3. de 13.3

elsewhere. In parallel, there is some, although inconstant, evidence that alkaline phosphatase, which is an integral constituent of the brush border membrane, may be involved in phosphorus transReceived for publication February 5, 1980 and in revised form November 5, 1980

0085-2538/81/0020-0181 $01.40

© 1981 by the International Society of Nephrology 181

Brunette et

182

port in some microorganisms [5—7] and also in mammals kidney [8—11].

In an attempt to elucidate the various factors that may influence phosphorus transport in vitamin-D-resistant rickets, we assessed the alkaline phosphatase activity along the nephron of rachitic mice (Hyp mice) and compared these values with the corresponding enzyme activity in normal mice. The results

demonstrate a significant decrease in alkaline phosphatase activity in the early proximal convoluted tubule of Hyp mice. Methods

Animals. All experiments were performed in male mice (strain C 57 BL-6J) that were either normal or rachitic with hypophosphatemia (Hyp). The mutant strain was originally provided by Drs. Eicher, Scriver, and Tenenhouse, and subsequently bred in our laboratory. Normal mice were usually from the same family as the rachitic animals. In a few instances, however, they were supplied by Charles River Laboratory Inc. (Wilmongton, Massachusetts). Results of the enzyme assay were not different between animals from the two origins. To make mice phosphorus-depleted, we fed normal mice for 7 to 14

days with a low phosphorus diet (I.C.N. diet no. 104671) containing 0.51% calcium and 0.03% inorganic phosphorus, thus rendering them hypophosphatemic. Microdissection. After i.p. anesthesia with mactin®, and

systemic injection of heparin, the animals were bled, the abdomen opened, and the left kidney perfused in situ with 1 ml of a mixture of collagenase (1 mg/ml) in Hanks' solution. The kidney was excised, and small pyramids of parenchyma were isolated from sagittal slices. These pyramids were incubated for 1 hour at room temperature in collagenase solution saturated with oxygen. Dissection was performed in cold Hanks' solu-

tion. The various fragments of the nephron were identified according to the description of Morel et al for the rabbit [12, 131,

and recently for the mouse [14] where the proximal tubule is further divided into an initial postcapsular segment of 1 mm length (S1), a mean segment of approximately 1 mm distant from S1 (S2), and the terminal part of the pars recta (S3).

Fragments isolated from the rest of the nephron were the descending limb (Desc), the medullary (MAL), and the cortical (CAL) ascending limbs of the loop of Henle, the bright (DCTb), granular (DCTg), and light (DCT1) portions of the distal tubule, and finally the cortical (CCT) and medullary (MCT) collecting tubule. The dissected segments were transferred to the depression of a bacteriology slide and photographed for subsequent length measurement. Cell disruption was achieved by a preincubation in 0.2 ii.l of a hypotonic solution for 30 mm and freezing on dry ice for a few seconds.

Solutions and compounds. Collagenase was obtained from

al

tubule was determined using a microadaptation of a technique described by Butcher and Lowry [15], which we previously reported [16].

Microassay of alkaline phosphatase activity. The alkaline phosphatase activity was determined according to a technique developed in our laboratory, using 4-methylumbelliferyl phosphate as a substrate. The fundamental reaction, as described by Cornish, Neale, and Posen [17], consists of an incubation of the phosphatase containing tissue with the substrate, which, upon dephosphorylation, becomes highly fluorescent. Because a number of modifications have been made to the original technique to adapt it to minute quantities of enzyme (a

few picomoles per sample), we summarize here the entire

procedure. The substrate is 4-Me umbelliferyl-phosphate (mol wt, 256). The stock solution (33 M) is prepared in buffer 1, and the working solution (6 p.M) in buffer 2. Buffer 1 consists of 57

m sodium bicarbonate and 42 mEq/liter sodium carbonate, with a final pH adjusted to 9.8. Buffer 2 is composed of 90 mi

sodium bicarbonate, 10 mEq/liter sodium carbonate, 4 mi magnesium sulfate, and 0.1% Triton. The final pH of buffer 2 is adjusted to 9.2. Standards are made with 4-Me umbelliferone (mol wt, 176.2). The stock solution of 4-Me umbelliferone (1 mM) is prepared in methanol, and the working solutions (12.5— 25—50 ILM) in buffer 2. Standard curve is prepared by adding 5 p.1 of each standard to 120 p.1 of substrate, working solution. The fluorescence is read in microcaps (Drummond) with the micro-

fluorometer AMINCO. Primary filter was 360 mp. and the secondary filter was 465 mp. in wavelength. Dosage of the samples. Each fragment of tubule is placed in 0.5 p.1 of hypotonic solution, in the depression of a bacteriology slide. Substrate (20 p.l) is deposited on the fragment. The slides are then tightly covered to prevent evaporation, and incubated in water for 30 mm at 35° C at 30-sec intervals. After incubation, the preparations were collected in microcaps, centrifuged, and fluorescence was immediately measured at 30-sec intervals.

The results, initially expressed as concentrations of 4-Me umbelliferone, are converted into absolute values contained in

the 20 p.1 of incubation medium, and finally reported per millimeter of tubular length, per 30 mm of incubation. The fluorescence obtained with this technique was found to be directly proportional to time up to 30 mm of incubation time, and directly proportional to the tubular length: y = —1.66 + 19.32x; r

= 0.81; N =

116.

To confirm the validity of the method, we made the comparison with the more classical p-nitrophenyl phosphate technique [18]. Twenty-six preparations of either kidney homogenates (N = 14) or brush border vesicles (N = 12) were simultaneously measured by the two techniques. The coefficient of correlation was 0.998, indicating that most probably both substrates were

Clostridium histolyticum (Sigma type 1) and diluted in modified

hydrolyzed by the same enzyme. Furthermore, whereas l-p-

Hanks' solution (1 mg ml') containing bovine serum albumin (1 mg mt'). The modified Hanks' solution contained 137 mrvi sodium chloride, 5.15 m potassium chloride, 0.80 m magnesium sulfate, 0.33 m dibasic sodium phosphate, 0.44 monobasic potassium phosphate, 1.05 mrvi 10 mivi Tris hydrochloride, and 1.0 m calcium chloride. The hypotonic solution consisted of 0.25 mr.t EDTA, 1.0 m magnesium chloride, and 8.0mM Tris hydrochloride. Bovine serum albumin (1 mg m1') was added.

bromotetramisol (1.0 mM) inhibited the dephosphorylation of 4-

Microassay of protein. The mean protein content of the

methylumbelliferyl phosphate at a pH of 9.8 by 96%, the ouabain had no significant effect upon the reaction (—2.06 1.63%,

N=

17).

Results

Normal mice. The profile of phosphatase activity along the mouse nephron was studied in 20 preliminary experiments. In each experiment, values consisted of the mean of three to five preparations of the various segments. Results are presented in

183

Phosphatase activity in rachitic mice

Table 1. Phosphatase activity along the proximal convoluting tubule in hypophosphatemic mice (Hyp) vs. normal micea .13

S3

pmoles of substrate/mm per 30 mm

>E:i

S Normal mice

lIE

,0. '

Hyp mice

.13

19.7

13.3

1.3

(N =

24)

(N =

15)

14.8

0.8

13.8

P

Cfl 4

Or.

-.3

(N = 24) <0.005

Values are the means

13

S

1.2

(N = NS

1.0 15)

0.5

0.1

(N = 5) 0.7 0.5

(N 5) NS

SEM. P values were obtained by the

Student's group t test.

13.

S1

S2 S Desc MAL CAL DCTb DCTg OCT1 CCI MCI

Fig. 1. Phosphatase activity along the nephron in 20 normal mice. S1

and S2 are early and late proximal convoluted tubules. S3 is pars recta.

tubular volume or protein content is not accurate either, because the enzyme is localized on one surface only of the cell. Therefore, to express alkaline phosphatase activity per microgram of protein would tend to overestimate the enzyme activity

Desc, MAL, and CAL are descending, medullary ascending, and cortical ascending limb of the loop of Henle. DCTb and DCT1 are for smaller mice. The persistence of a low enzyme activity in bright, granular, and light segments of the distal convoluted tubules. rachitic mice despite such an artificial overcorrection of results CCT and MCT are cortical and medullary collecting tubules. The should confirm the abnormal finding. phosphatase activity is expressed as picomoles of dephosphorylated substrate (4-methyl umbelliferyl-phosphate) per millimeter of tubule per 30 mm of incubation.

Fig. 1. They are expressed in picomoles of hydrolyzed substrate per millimeter of tubular length per 30 mm of incubation. High activities were found in segments S1 and S2: 20.3 (sEM) 1.04 1.1, respectively. The two values are significantly and 13.8 different from each other (P < 0.001). Phosphatase activity was

also found in the glomeruli (not mentioned in Fig. 1), with a mean value of 1.28

0.08 pmoles/glomeruli. None of the other

segments, including S3, revealed any significant amount of enzyme activity. Trace activity was repeatedly found in DCTb and DCTg, but the mean values were not significantly different from the control levels observed in the dissecting medium. Rachitic vs. normal mice. The experiments were performed

In a series of 15 normal mice of various weights, tubular protein per millimeter of length was measured and plotted against the body weight of the animals. Fifteen to twenty millimeters of dissected tubules were required for each measurement, and each value reported corresponds to the mean of three or four measurements in one animal. Because separation

of S2 from S1 is fastidious and the two segments have an identical diameter under microscope, no distinction was made between them. As expected, there was a significant relation between the protein content and the body weight: y = 8.66 + Q.5x; N = 15; r = 0.67; P < 0.005. The addition of 9 rachitic mice and 4 animals depleted of Pi did not modify the regression line nor the P value. This relation was thus used to calculate the phosphatase activity per microgram of tubular protein in each

experiment comparing Hyp vs. normal mice. The detailed results are presented in Fig. 2, expressed as picomoles of

on 24 rachitic, paired with 24 normal mice. Each day, both

dephosphorylated substrate per millimeter of tubular length on

rachitic and normal mice were prepared and dissected together,

the left, and per microgram of tubular protein on the right. Again, the phosphatase activity was found to be depressed in Hyp mice (77 4.2 vs. 94.8 5.4 pmoles/g protein in Hyp

and alkaline phosphatase activities were measured simultaneously to minimize day-to-day technical variations. Results were analyzed statistically using the Student's t group test. Because no enzyme activity was found in the distal segments of

either series of animals, only those results obtained in the

and normal mice, respectively (P < 0.02).

To investigate a possible age factor, we plotted enzyme

activities in S1 segments (pmoles/J.Lg of protein per 30 mm) proximal segments are presented (Table 1). The phosphatase against the age in the two series of mice (Fig. 3). The tendency activity in S1 is significantly lower in Hyp compared with the for the Hyp mice to have low values of phosphatase is again normal mice: 14.8 0.8 and 19.7 1.2 pmoles/mm per 30 mm, apparent. In Hyp mice, there is no evidence of any correlation respectively (P <0.005). On the other hand, no such differences between the two variables. In normal mice, a certain positive slope of 0.43 observed (0.61 if the late value is dropped), which were observed in S2 nor S3. Because the Hyp mice are smaller than the normal animals does not reach, however, the criteria for statistical significance. are, the mass of tubular parenchyma per millimeter of tubular To minimize the problem of size differences, Hyp mice were length should vary with the total weight of the animal, and usually chosen slightly older than their paired controls. Considdifferences between the results obtained in the two series of ering the lifespan of several years, however, all these mice were mice could depend on the way they are expressed rather than relatively young. If in normal mice, alkaline phosphatase has a representing a true variation of enzyme activity. In other tendency to increase with age, the finding of a low enzyme words, rachitic mice may present less alkaline phosphatase per activity in the rachitic mice despite their slightly older age millimeter of tubular length because their nephrons are smaller. appears still more significant. Finally, the vitamin-D-resistant rachitic mice with hypophosBecause alkaline phosphatase is essentially localized in the brush border membrane, that is, on the surface, the proper way phatemia are Pi depleted. To compare these mice with normal to report its activity should be per unit of brush border surface, animals similarly depleted of Pi, we measured the enzyme in an which is not feasible. To report the enzyme activity per unit of additional series of 14 Hyp mice, paired with normal mice

Brunette et

184

Hyp Normal

Normal

al

Hyp

• Normal V 70.8 + 0.43X

180

Hyp Y66.0+0.12X

160 140

25'

g

-120

1-'

.100 o,

2° °-

.o

> 20'

0

-80

15.

120 100 80 60

40

0. 20

30 40 50 60 70 80 90 100 110 120 130 140 Age, days

Fig. 3. Phosphatase activity in S1 as a function of age in normal and

.60

10

Mean = 19.7 SD SCM

= 5.9 = 1.2

N

=24

14.8 3.8 0.8 24

94.8 26.3 5.4 24

77.0 20.4

4.2 24

hypophosphatemic (Hyp) mice. The relations are not significant, suggesting that between 30 to 140 days of age, the enzyme activity per microgram of tubular protein remains constant.

convoluted tubule, particularly in the early part, that is, S1. The terminal part of S3 and the following segments of the nephron are devoid of any significant activity. Longley and Fisher [19]

and Hardonk and Koudstaal [20] using histochemical tech-

niques, have studied alkaline phosphatase activity in the developing kidney of the mouse. They reported that during the first Fig. 2. Phosphatase activity in S1, in paired hypophosphatemic (Hyp), days of life, enzyme activity was present in some proximal and normal mice. Results are expressed as picomoles of dephosphorytubules only, and probably at their distal part. After 8 days, lated substrate per unit of tubular length on the left, and per micrograms of tubular protein on the right side of the figure. In both modes of activity is present through the whole kidney. After 3 weeks, presentation, enzyme activity is significantly depressed in Hyp mice, there is a strong decrease in "P2" segment, which corresponds compared with the corresponding values in normal mice (t group test). to S3, and in adult mice, the enzyme activity in "P2" segment is completely absent. All of our experiments were performed in animals of 5 weeks or more. The lack of phosphatase activity in depleted of phosphorus for 7 to 14 days. Table 2 represents S3, as well as the relative constancy of this activity in S along clearance data in normal, Hyp, and normal Pi-depleted mice. the range of weight and age covered by our experiments, Plasma phosphate was comparable in Hyp and normal Pi- confirms the findings of Hardonk et al in mice. But, species depleted (1.83 0.08 and 1.89 0.16 mmoles/liter, respective- differences are possible, because Jacobsen, Jorgensen, and iy, compared with 2.84 0.1 in mmoles/liter in normal mice Thomsen [21] reported a high enzyme activity in the pars recta and the urine-to-plasma phosphate was in the same range in of the rat. Hyp and normal mice (38.6 6.4 and 30.9 As far as the rest of the nephron is concerned, we are not 5.7, compared with 0.6 0.3 in normal Pi-depleted mice). Plasma calcium aware of any study performed in the mouse. Gomori [22] concentration showed a slight tendency to decrease in Hyp, and studied nine species, and reported that the glomeruli in all to increase in normal Pi-depleted mice. species were negative, the proximal convoluted tubule (PCT) The results of alkaline phosphatase activities in S1 in this last positive, as well as a portion of varying length of the pars recta. series of experiments are shown in Fig. 4. Regardless of the way In man, the ascending limb of the loop of Henle was also the results are presented, per unit of tubular length or protein positive. The distal and the collecting tubules were negative in content, it is evident that the low activity of phosphatase in Hyp all species. Wachstein [23] was not able to confirm the activity cannot be explained by Pi depletion that increases rather than in the ascending limb of man, and few years later Bonting et al decreases the enzyme activity. The value of 120.7 5.3 pmoles [24, 25] increased the controversy by reporting the presence of of Pi per microgram of protein in normal Pi-depleted mice not enzyme activity along the whole nephron of man. In all studies, only is significantly different from 72.0 4.0 found in Hyp (P < however, the highest levels of alkaline phosphatase are consis0.001), but is also different from 94.8 5.4 previously reported tantly found in the PCT, and more specifically in the brush in the study of normal mice (P < 0.005). border membrane [21, 22, 26, 27] where the enzyme constitutes, In contrast, no difference in phosphatase activity could be at least in man, an integral part of the membrane structure [28]. demonstrated in S2 of the two series of experiments (Fig. 5). Alkaline phosphatase activity in Hyp mice: (1) A primary But, the lack of reproducible results, which might be attributed disorder? The alkaline phosphatase activity was found to be to the difficulty to recognize comparable segments S2 among relatively low in the S segment of our Hyp mice. It is also in proximal tubules of various length, may have masked any this segment that the transport defect in Hyp mice was found difference between Hyp and normal, or Hyp and normal Pi- [2]. It is tempting to hypothetize a relationship between these depleted series of animals. two observations. The decrease of enzyme activity may be the primary phenomenon or, on the contrary, a more or less direct Discussion consequence of the Pi transport defect. It may reflect a primary Phosphatase activity along the normal nephron of male mice. defect of synthesis of a normal molecule, or the synthesis of an Alkaline phosphatase is essentially localized in the proximal abnormal enzyme. If several isoenzymes exist, which has been P <0.005

P <0.02

185

Phosphatase activity in rachitic mice Table 2. Clearance dataa

Plasma phosphate mmoles/liter 2.84

Hyp

1.83

Normal phosphate-depleted

1.89

4.69

5.7 (N = 18)

0.10 (N = 24)

Normal

Plasma calcium mEqiliter

Urine-to-plasma phosphate 30.99 38.60

0.08°

4.28

6.42

0.64

0.16°

5.39

0.30°

0.18"

(N = 14)

(N = 14)

(N = 14)

0.26

(N = 12)

(N = 19)

(N = 27)

0.22

(N = 10)

a Values are the means SEM. Hyp is hypophosphatemic mice. Phosphate values are for inorganic phosphate. P < 0.02 (vs. normal). P < 0.001 (vs. normal).

Normal Hyp

Hyp

Phosphate-depleted

140

-140

110

-110

depleted Hyp

.150

80

0.0.

0.

0.

-50

50

30-

>

: -80 '

:1

-8

Phosphatedepleted

Phosphate-

Hyp

2 0

.100

20E

0.

.50 10-

20 Mean so SCM

N

-20 = 70.5

66.5

61.7

79.8

=20.8

25.1 6.5

17.4

32.1 8.6 14

= 5.4 =15

15 MS

4.7 14 NS

Fig. 4. Phosphatase activity in S, in hypophosphatemic (Hyp) paired to phosphate-depleted mice. Results are expressed as picomoles of dephosphorylated substrate per unit of tubular length on the left, and per microgram of tubular protein on the right side of the figure. The enzyme activity is significantly lower in Hyp mice compared with the corresponding value in normal mice who are phosphate-depleted (t group test).

Mean so

= 14.4 3.2

SEM

14

N

= 0.9

24.6

72.0

4.7

15.1

1.2 14

4.0 14

120.7 19.7 5.3 14

p<0.001 p<0.001 Fig. 5. Phosphatase activity in S, in Hyp mice paired to normal (on the left) and phosphate-depleted (on the right) mice. No significant difference of enzyme activity has been observed in either of the two groups of experiments.

ion is concerned, no consensus exists. Although some studies deny any influence [30, 36, 371, some others report a reproduc-

ible increase of the renal alkaline phosphatase activity in Pidepleted rats, an increase that is curtailed by the administration

recently suggested [291, only one isoenzyme may be involved. Km determination in cortex homogenates of normal and Hyp of actinomycin [8—10, 38]. Our data confirm in mice the second mice have been performed in another series of experiments, opinion, at least in the segment S1. Thus, the chronic depletion which are not reported here. There was no difference between of Pi that is a trait of the disease, far from explaining the low the mean values obtained in the two groups of animals, suggest- enzymatic activity in rachitic mice, rather reinforces the eviing a quantitative rather than a qualitative disorder of the dence of the defect. (3) Alkaline phosphatase and P1 transport. As long as the enzyme. But the fact that alkaline phosphatase is present only in one segment of the nephron does not validate negative results relationship, if any one exists, between alkaline phosphatase

obtained in the entire cortex homogenate, particularly if the possibility of one isozyme defect is considered. (2) A secondary disorder? The hypothesis of a decreased enzyme activity secondary to another factor is also plausible. But the nature of this factor would be totally obscure. Physiologic factors that regulate alkaline phosphatase activity are unknown and those that regulate Pi transport do not systematically influence the enzyme activity. In fact, it seems that PTH, and 1 ,25(OH)2D3 have no influence upon the brush border alkaline phosphatase [30, 31, 36], and moreover, the circulating

and Pi transport is unknown, interpretation of our data will remain hypothetical. As already mentioned, regulating factors for Pi transport do not systematically modify alkaline phosphatase activity. Conversely, pharmacologic inhibition of the enzyme does not systematically modify Pi transport either. Levamisol in vivo seems to simultaneously inhibit alkaline phosphatase and Pi transport [11], but EDTA and Levamisol in vitro do not affect Pi transport through brush border vesicles, despite decreasing enzyme activity by 50% to 90% [30, 391. The lack of consistency between alkaline phosphatase activity and Pi transport, however, does not necessarily eliminate any hypothetical

PTH and I ,25(OH)2D3 concentration in vitamin-D-resistant rickets are normal [1, 33—35]. As far as the phosphorus deple- link between them. Indeed, it is possible that Pi transport

Brunette et a!

186

results from a multitude of factors, the alkaline phosphatase being one among these factors; it is also possible that alkaline phosphatase is indirectly modified by Pi transport, or that only some of the numerous factors that affect Pi transport also influence the enzyme activity without any direct link between

11. PLANTE GE, ERIAN R, NAWAR T, PETITCLERC C: Renal transport

the two phenomena.

13. MOREL F, CHABARDES D, IMBERT M: Functional segmentation of

(4) Vitamin-D-resistant rickets: A complex disease. It is evident that the data presented here do not solve the problem of the pathogenesis of the hypophosphatemic vitamin-D-resistant rickets. Rather, they constitute another observation whose link with previously accumulated data is not evident. These rachitic

mice partially respond to parathyroidectomy [40] and to Pi depletion [32] by increasing Pi tubular reabsorption. Their adenylate cyclase response to PTH in the proximal tubule is decreased by half, and to calcitonin in the distal tubule multiplied by a factor of six [14], despite normal circulating levels of these hormones. Finally, a relative defect in alkaline phosphatase activity is reported in the present study.

As long as the physiologic interrelationship between all factors regulating Pi transport remains obscure, the integration of these accumulated observations will remain a hypothetical exercise. Acknowledgments This work was supported by National Institutes of Health grant AM25615-02 and Medical Research Council grant MT-4472. Dr. S. O'Regan contributed in the preparation of the manuscript.

Reprint requests to Dr. M. G. Brunette, Maisonneuve-Rosemont Hospital, Research Center, 5415 I'Assomption Blvd., Montreal HIT 2M4 P.Q. Canada

References I. EICHER EM, SOUTHARD JL, ScRIvER CR, GLORIEUX FH: Hypophosphatemia: mouse model for human familial hypophosphatemic

(vitamin D resistant) rickets. Proc Nat! Acad Sci USA 73:4667— 4671, 1976 2. GlAssoN SD, BRUNETTE MG, DANAN G, VIGNEAULT N, CARRIERE S: Micropuncture study of renal phosphorus transport in hypophos-

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