nism for renal threshold for glucose - Journal of Biological Chemistry

KIDNEY AND

PHOSPHRTASE IN ALIMENTARY HYPERGLYCEMIA PHLORHIZIN GLYCOSURIA. A DYNAMIC MECHANISM FOR RENAL THRESHOLD FOR GLUCOSE BY JULIAN WITH

(Prom

13. MARSH

AND DAVID

THE COLLABORATION

the Depcwtmenls Graduate School

of

OF WILLIAM

I-'h~~siolo~ical

of Medicine,

University PLATE

(Received

for publication,

L. DRABKIN B. GODDARD

Chemistry, School of Pennsylvania,

of Medicine

and

the

Philadelphia)

1 September

27, 1946)

1 Personal

communication

to ono of us (D. 1,. D .) from Al

E. Lundsgnartl.

Downloaded from http://www.jbc.org/ by guest on November 25, 2017

Glucose, which appears in the glomerular filtrate at all levels of blood sugar, is reabsorbed mainly in the proximal convoluted tubules of the kidney. This has been established by Walker and Hudson (1) by means of micro dissection and microanalytical techniques. Under usual physiological conditions, the reabsorption of the sugar is complete. The renal threshold for glucose may hence be defined as that level of blood glucose beyond which complete tubular reabsorption no longer occurs. Involvement of the phosphorylation process in the mechanism of reabsorption of glucose was postulated by Lundsgaard and his colleagues (2, 3) in the explanation of phlorhizin glycosuria. Beck (4) studied the effect of phlorhizin on phosphorylation and dephosphorylation, and partially confirmed Lundsgaard’s views, although Beck’s positive findings w&h reference to dephosphorylation were limited to acid phosphatase. It is of interest that histochemical staining techniques (5, 6) suggest that the proximal tubules are one of the preferential sites of high concentration of the kidney phosphatase. Recent reviewers (7, S), on the other hand, have regarded the evidence for the r81e of phosphatase in tubular glucose reabsorption and particularly the inhibition of kidney phosphatase by phlqrhizin as entirely inadequate. It should be pointed out that the work of Lundsganrd and his colleagues (2, 3) and Beck (4) has been confined to experiments in vitro. As far as we know, a demonstration of the relation of phosphorplat,ion-dephosphorylation in glucose reabsorption in kidney tubules has not, been demonstrated in uiv0.l One of the difficulties in earlier work is the confusion which has resulted from a lack of distinction between phosphorylation and dephosphorylation. It is now recognized that, under the conditions existing in tissues, the position of equilibrium is such that the catalysis by phosphatase is essentially non-reversible. Phosphatase catalyzes only the dephosphoryla-

62

PHOSPHATASE

AND

GLYCOSURIA

Methods Extraction of Phosphatase-Albino rats of both sexes, 200 to 500 gm. in weight, fasted for 24 hours, were used. The kidneys were secured by unilateral or bilateral nephrectomy, rapidly performed under moderate ether anesthesia. After as much connective tissue as possible was quickly removed, and the excess blood blotted with filter paper, the kidney was frozen with liquid air and ground to a powder in a mortar. This is essentially the procedure of Perlmann and Ferry (9). In preliminary studies of extraction procedures we have found that homogenization of the above powder results in higher phosphatase activity (appreciably higher than that reported for kidney by Beck (4)). An addibional step was accordingly introduced: 0.2 to 0.3 gm. of the powder, representing approximately onefifth of the original rat kidney, was transferred to a weighed test-tube homogenizer of the Potter-Elvehjem type (10). The tube was reweighed,


tion of certain phosphate esters. The phosphorylation process is biologically under the control of separate, complex enzyme systems involving hexokinase and adenosine triphosphate (ATP). It should be understood, therefore, that studies of phosphatase are directed mainly towards However, as will be seen later, in biologithe dephosphorylation reaction. cal systems the separation of the dephosphorylation and phosphorylation In the present investigation of the possible r&e process may be difficult. of phosphorylation-dephosphorylation in the renal threshold for glucose, attention has been directed to a study of phosphatase activity in fwo conditions: ihduced alimentary hyperglycemia with glycosuria and phlorhizin glycosuria. The study of the hyperglycemic state was undertaken, since it may be regarded as more physiological than phlorhizin poisoning. It was postulated that in acute hyperglycemia the tubular cells would be working at a maximum to reabsorb glucose, and, therefore, involvement of the phosphatase mechanism might be susceptible of demonstration. An improved method for the quantitative preparation of phosphatase It has been discovered from such extracts of kidney haa been developed. extracts that hyperglycemia induces a significant. increase in the level of both acid and alkaline phosphatase activity. This unexpected and interesting phenomenon received confirmatory evidence for its existence by a semiquantitative application of Gomori’s histochemical staining technique (5). Moreover, it has been clearly demonstrated that the addition of phlorhizin inhibits markedly both the acid and alkaline phosphatase activity of the kidney extracts, and that the phosphatase activity of phlorhizinized rat kidneys is diminished. As an aid in interpretation, data have also been gathered on certain aspects of the phosphorylation process in kidney.

J.

B.

MARSH

AND

D.

L.

DRABKIN

63


10 ml. of 0.5 M NaCl added, and the material homogenized for 2 minutes at room temperature. In the experiments involving phlorhizin greater concentration was required, and hence approximately 0.5 gm. of kidney powder was homogenized with 1.5 ml. of 0.5 M NaCl. Determination of Phosphatase Activity-Except in the phlorhizin experiments, 1 ml. of the homogenate was added to 9 ml. of a buffered sodium /3-glycerophosphate solution, prepared according to Shinowara, Jones, and Reinhart (II), and previously adjusted to 37.5”. In the experiments with phlorhizin, acid phosphatase activity was measured with 0.5 ml. of the concentrated homogenate and 1.0 ml. of the buffered substrate (Shinowara et al.), pH 4.9 to 5.1, while the determination of alkaline phosphatase required special conditions. 0.5 ml. of the concentrated homogenate was added to 1.0 ml. of a solution containing 5.0 gm. of sodium /3-glycerophosphate and 4.24 gm. of sodium diethyl barbiturate per 100 ml. The mixture was then adjusted to pH 9.2 by addition of 0.5 N acetic acid. The mixture of kidney extract and buffered substrate was incubated in a water thermostat. for 1 hour at 37.5”. 5 ml. of 10 per cent trichloroacetic acid were then added to inactivate the enzyme. In each determination of phosphatase activity, aliquots, in which the enzyme was. inactivated by addition of trichloroacetic acid at the start of incubation, were used as controls to correct for the inorganic phosphate originally present in extract and substrate. Both the experimcnt,al and control solutions were filtered after inactivation. The inorganic phosphate present in an aliquot (usually 2 ml.) of each filtrate was determined by the method of Fiske and Subbarow (12), with a Klett-Summerson (13) photoelectric photometer with the red (X 660 mp) filter. The pH of the buffered sodium /3-glycerophosphate solutions was checked by a glass electrode. Blood sugar was determined by the micromethod of Folin and Malmros (14), also by means of the photoelectric photometer but with the green (X 540 mp) filter. Determination of Adenosine Triphosphate-O.5 to 1.0 gm. of powdered frozen kidney tissue was added to 7 ml. of 7 per cent, trichloroacetic acid in a tared centrifuge tube at 0”, according to the procedure of Kaplan and Greenberg (15). The 7 minute-hydrolyzable phosphate was determined directly on the trichloroacetic acid filtrate made up to a volume of 25 ml. Determination of Aerobic Phosphorylation-The method used was essentially that of Colowick, Welch, and Cori (16). , Both oxygen consumption by Warburg’s direct manometric technique (17) and decrease in inorganic phosphate were measured. The kidneys were removed from decapitated rats and immediately placed on cracked ice, weighed, and transferred to a homogenizer tube kept in ice. Sufficient 0.05 M phosphate buffer of pH 7.5 was added to make the concentration approximately 180 mg. of tissue per 1 ml. of homogenate. The material was homogenized for 1 minute.

64

PHOSPH.4TASE

AND

GLYCOSURIA

EXPERIMENTAL

The data collected in Tables I and II indicate that alimentary hyperglycemia was accompanied by a statistically significant (18) increase in the acid and alkaline phosphatase activities of rat kidney homogenates. The increase amounted to 55 per cent for the acid and 70 per cent for the alkaline phosphatase. That this rise was not accounted for by dehydration of the kidney tissue as a consequence of the administration of hypertonic glucose to a fasted animal was evident from the constancy of the wet weight-dry weight ratios. As an independent check on these results, kidney sccti0n.s from a fasted and from a hyperglycemic rat were stained for acid phosphatase by the Gomori t.echnique (5). By limiting the incubation period to only 7 hours at 37.5”, it was possible to make a semiquantitative demonstration by this In Fig. 1, A, the tissue from the fasted rat showed only very method. slight staining for “phosphatase activity,” whereas in Fig. 1, B, the kidney section from the hyperglycemic animal was stained very heavily. In Table III we present data showing that phlorhizin added in vitro was capable of inhibiting markedly both acid and alkaline phosphatase activity of rat kidney extracts. The magnitude of the inhibition depended on the phlorhizin concentration. In contrast, with the findings of Beck (4), in our experiments the effect was more pronounced in the pH range of alkaline phosphataee. Evidence that the inhibitory effect of phlorhizin on kidney phosphatase activity can also be demonstrated in uivo is furnished in Table IV. We could not demonstrate the effect of the drug when we measured the activity of the dilute kidney homogenates from phlorhizinized rats. In the successful demonstration, the concentrated homogenates previously described,


1 ml. aliquots of this homogenate were then added to the Warburg vessels containing 1 ml. of substrate and 0.2 ml. of 2 N NaOH in the center well. The composition of the substrate per ml. was glucose 10 mg., NaF 3 mg., magnesium citrate 0.2 mg., and Z(+)-glutamic acid 4 mg., adjusted to pH 8.0 with 1 N NaOH. The vessels were gassed with 100 per cent oxygen for 2 minutes, and equilibrated at 38.3” for 8 minutes. Oxygen consumption was then measured for the next 10 minutes. After 30 minutes, the vessels were disconnected from the manometers and 2 ml. of 7 per cent trichloroacetic acid were added. The contents were transferred to 100 ml. volumetric flasks, brought to volume, and filtered. The inorganic phosphate was determined on aliquom of the filtrate. At the start of the incubation period trichloroacetic acid was added to one of the vessels, the filtrate of which was used for the measurement of the inorganic phosphate originally present in homogenate and substrate.

J.

B. MARSH

AND TABLE

Acid

Phosphatase

D. L.

65

DRABKIN

I

Activity,

at pH 4.9 to 5.1, per Gm., Wet Weight, 24 Hour-Fasted and Hyperglycemic Rats

of

Kidney

Tissue

in

-

state

Rat No.

Glucose dosage* gm.

163 17§

18§ 198 20 21 22 23 24 25 27 28 29 30 35 37 9 10 11 12 13 14 15

16s 17s

l8§ 1% 26 31 32 33 34 46 47

Time of analysisi

Blood S”&U

bs.

mg. per

Fasted I‘ I‘ I‘ “ “ “ “