Pflfigers Archiv

PflfigersArch. 371, 33- 38 (1977)

EuropeanJournal of Physiology 9 by Springer-Verlag1977

Micropuncture Study of Renal Phosphorus Transport in Hypophosphatemic Vitamin D Resistant Rickets Mice SUZANNE D. GIASSON*,MICHELEG. BRUNETTe!. GABYDANAN, NORMANDVIGNEAULT,and SERGE CARRIERE with the technicalassistanceof LOUISFAFARD Laboratoryof Electrolytes,MaisonneuveHospital, Universityof Montreal,Schoolof Medicine,MontrealP. Q., CanadaHIT 2M4

Summary. A micropuncture study of inorganic phosphorus (Pi) transport was performed in 6 mice presenting hypophosphatemic vitamin D resistant rickets (Hyp) and results compared to those obtained in 13 normal (N) mice. The mean plasma (P Pi) and fractional excretion of Pi (FE Pi) in N and Hyp mice were 85.10 + 2.27 rag/1 and 15.81 -t- 1.90 vs 48.43 -t- 3.29 rag/1 and 35.34 __+ 5.26 ~, respectively. In N mice, tubular fluid over plasma Pi ratio (TF/P Pi) progressively decreases along the proximal tubule to reach approximately 0.6 in the late accessible part. The fraction of filtered Pi ( ~ E Pi) remaining in this segment of the nephron is therefore 2 0 - 2 5 ~ . In Hyp mice the TF/P Pi in proximal tubule remains relatively high and does not significantly vary with TF/P inulin (mean TF/P Pi = 1.19 _+ 0.12 S.E., compared to 0.73 _+ 0.04 in N mice). Precise conclusions concerning ~ E Pi at the end of the proximal tubule of Hyp mice are not available because of the scattering of the data. However, all the values of ~ E Pi (mean E Pi' 78.68 __ 7.39 compared to 49.10 -t- 4.59 in N mice) are far above the total urinary FE Pi (35.34 + 5.26), suggesting a relatively greater distal fractional Pi reabsorption in Hyp mice than in N mice. Since the P Pi, and therefore the amount of filtered Pi is lower in Hyp mice, it is probable that the absolute amount of Pi reabsorbed distally is comparable in the two series of animals. Key words." Renal electrolytes - Phosphorus transport - Vitamin D resistant rickets phatemia.

Hypophos-

This work was supported by the MedicalResearchCouncilof Canada, grant MA 4472 and by specialfunds fromthe University of Montreal * Researchfellowin renalphysiology,supported by the conseilde la rechercheen sant6 du Quebec

INTRODUCTION Since the first description of hypophosphatemic vitamin D resistant rickets (Hyp VDRR) by Albright et al. [3], the pathogenesis of this hereditary disease is still unknown. Two main hypothesis are in opposition regarding the primary defect responsible for the constant hypophosphatemia: The first suggests a faulty intestinal absorption of calcium, with secondary hyperparathyroidism [3,17] whereas the second one puts the initial responsibility on a defect of renal transport of inorganic phosphorus (Pi) [15,23,43]. The latter hypothesis is supported by the observation of Pi secretion during systemic phosphorus loading in these patients [23,43]. The measurement of the serum immuno-reactive parathyroid hormone, however, has been found either low [37] normal [6] or elevated [29, 36]. Until recently, the exclusive incidence of the VDRR in human being greatly reduced the possibilities of investigation, and our information concerning Pi transport at the cellular level could not be fully utilized to the understanding of the disease pathogenesis. The recent discovery, by Eicher and collaborators (Jackson Laboratory, Bar Harbour, Maine U.S,A.) [16] of a mutant strain of mice exhibiting hypophosphatemic vitamin D resistant rickets will provide in a near future, an opportunity to get new information upon the disease, by performing studies of calcium and phosphorus transport in various tissues of these mice. The present report concerns a micropuncture study of Pi tubular reabsorption in normal and in mutant mice. Results indicate that Pi reabsorbtion in proximal tubule is decreased in mutant mice. In contrast, an important fraction of the filtered Pi is reabsorbed distally.

34

Pfliigers Arch. 371 (1977)

METHODS Twenty-one punctures have been preformed in 13 control (C 57 BL/6 J) male mice (20.7 • 3.4 g) and 14 in 6 hypophosphatemic male mice (C 57 BL/6 J Hyp) (20.0 • 1.7 g). The animals were anesthetized with inactin, 120 mg/K IP. Catheters were placed in the carotid artery and the jugular vein for blood sampling and intravenous perfusion. Urine was collected through a bladder catheter. Following a priming dose of inulin given in Ringer-lactate (0.4 ml in 30 min), a maintenance infusion (0.013 ml/min) was given in order to keep the plasma inulin concentration at approximately 30 rag/100 ml. The left kidney was then prepared for micropuncture through an abdominal incision, and proximal convoluted tubules punctured through the capsule. Inulin and Pi clearances were performed every second hour, during the time of micropuncture. The punctures were performed with sharpened pipettes 5 - 6 um OD, and the mean volume collected approximated 10 nl.

in control mice, but since UV is increased in the first group, the phosphaturia is comparable in the two series of animals, (1.12 + 0.22 and 1.79 + 0.31 gg/ min in Hyp and N mice, respectively). However, due to the different P Pi, the FE Pi is largely increased in Hyp (35.34 +_ 5.26 ~) compared to the corresponding value in N mice (15.81 4- 1.90~).

TF/pPi

1.0

0.8

Analytical Methods. Inulin concentration in plasma and urine was measured by the photometric technique of Heyrovski [24] and inorganic phosphorus was assayed according to the technique of Chen and Toribara [12]. Tubular fluid inulin was measured using the fluorometric method of Vurek and Pegram [41]. The intra tubular Pi concentration was determined by a new procedure recently developed in this laboratory. This technique is based on the reaction between phosphate, converted to h e x a d i molybdatophosphate, and thiamine, resulting in a highly fluorescent thiochrome. The microadaptation of this reaction permits measurement of Pi in 3 nl samples of tubular fluid. These samples, as the standards, were diluted 50 ~ in distilled water, added to the first reagent (ammonium molybdate and thiamine in H2SO4) which was secondarily mixed with the second reagent (Borax). The resulting fluorescence was read on the microfluorometer AMINCO, with a primary and a secondary filters corresponding to the excitation and the emission wave-lengths of the reaction (368 nm and 440 nm respectively). Pi determination in 50 plasma'ultrafiltrates and 17 urines by this microtechnique and by the macrotechnique of Chen and Toribara demonstrated an excellent correlation between the two techniques (R = 0.940 and 0.996, respectively) [10].

RESULTS

0.6

i

~

t 9 ..4L_~ e oo

9

9

0.4

0.2

0.0 1.0

,

i

1,2

1.4

1.6

1.8

i 2.0

= 2,2

2.4

2.6

T F / p I n. Fig. 1. Control mice. Micropuncture of the proximal tubules. Variations of TF/P Pi as function of TF/P inulin. The dotted line corresponds to the regression line

%EPi 100

80

!"

.;

20

The clearance data are presented in Table 1. P Pi in hypophosphatemic mice is 48.43 _+ 3.29 mg/1 compared to 85.10 + 2.27 mg/1 in normal mice. The G F R is also significantly lower in Hyp than in control mice, despite the fact that both groups' weight is comparable. Pi concentration in urine is lower in Hyp mice than

9

e~"

100

90

80

70

60

50

40

30

20

% E H20 Fig.2. Control mice. Micropuncture of the proximal tubules. Variations of ~ E Pi as function of ~ E H20. The dotted line corresponds to the regression line

Table 1. Clearance data Type of mice

P Pi mg/1

U Pi pg/pl

V pl/min

UV Pi Ixg/min

GFR p.1/min

C1 Pi gl/min

FE Pi

Control n = 21 (13 mice)

85.10 __ 2.27

4.30 • 0.53

0.41 • 0.05

1.79 • 0.31

132.71 _ 12.90

21144+_ 3.86

15.81 -t- 1.90

Hyp. n = 14 (6 mice)

48.43 • 3.29

1.74 • 0.20

0.68 • 0.14

1.12 • 0.22

90.64 • 14.88

24.29 • 4.11

35.34 • 5.26

Significancy

P < 0.0005

P < 0.05

P < 0.025

NS

NS

P < 0.0005

P < 0.025

S. D. Giasson et al. : Phosphorus Renal Transport in Resistant Rickets

35

TF/pPi 2.0

%[ Pi

1.8

t20

1.6 lOO 1.4 80 1.2 60

1.0 40

0.8 20

0,6 1.0

1.2

1.4

1.6

1.8

2,0

2.2

2.4

100

TF/p I n.

Mice

1 1 2 3 4 4 4 4 4 4 5 5 5 6

BO

70

60

50

40

30

% E H20

Fig.3. Hyp mice. Micropuncture of the proximal tubules. Variations of TF/P Pi as function of TF/P inulin. The dotted line: regression line of the corresponding values obtained in normal mice

Table 2.

90

Fig.4. Hyp mice. Micropuncture of the proximal tubules. Variations of ~ E Pi as function of ~ E H20. The dotted line: theoretical line corresponding to absence of Pi reabsorption

Hypophosphatemic mice. Detailed micropuncture data Phosphorus

Inulin

TF (rag/l)

P (rag/l)

TF/P

39 41 63 63 32 40 32 42 42 42 99 104 105 62

50 53 36 63 37 37 38 39 43 43 53 53 53 80

0.78 0.78 1.75 1.00 0.86 1.08 0.84 1.07 0.98 0.95 1.87 1.96 1.93 0.78

TF mg ~

P mg

TF/P

40

40 50 36 156 25 31 34 38 55 56 63 61 60 79

1.00 2.30

84 258 34 53 59 48 120 91 102

The micropuncture data are presented in Figures 1, 2, 3, and 4, and Tables 2 and 3. In control animals, the TF/P Pi ratio progressively decreases along the proximal tubule. Figure i represents the correlation between TF/P Pi (y) and TF/P In (x). The regression line (y = 0.98-0.15 x) is significant (R = -0.46, 0.01 < P < 0.05). The TF/P Pi in the late accessible portion of the proximal tubule approximates 0.6. Figure 2 shows the relationship between the fraction of filtered Pi remaining into the lumen ( ~ E Pi = y) and the fraction of unreabsorbed water ( ~ E H20 = x). Here again, the correlation between these two variables is significant (y = -0.12 +0.90 x R = 0.89, P