INVESTIGATIONS IN ENZYMATIC HISTOCHEMISTRY

III. DISTRIBUTION OF ENZYMES IN RABBIT KIDNEY

BY LEOPOLD WEIL AND ROBERT K. JENNINGS

(From The Biochemical Research Foundation of the Franklin Institute, Newark, Delaware)

(Received for publication, January 27, 1941)

The arginase activity of rabbit kidney was shown, in a previous paper (I), to be specifically associated with the cells of the proximal convoluted tubules. This investigation in enzymatic distribution has now been extended to cathepsin, aminopolypeptidase, di- peptidase, esterase, and amylase. The results of this study demonstrate that histological variations within the structure of the kidney are correlated with variations in the content of certain enzymes.

In the past, investigations concerning the functions of various tubule cells in the kidney were conducted primarily from the physicochemical point of view; it is hoped, however, that an enzymatic approach to this problem may contribute to a better understanding of the subject.

EXPERIMENTAL

Before the actual histoenzymatic work could be undertaken, it was necessary to make a study of those kidney enzymes which were to be investigated. Since the literature yielded very little information concerning the proteinase of kidney, preliminary research into the character of this enzyme was necessary.

The pH optimum of kidney proteinase was measured; clupein and edestin were used as substrates. In the case of clupein sulfate, the progress of the enzymatic breakdown was followed by micro form01 titration (2), while in the case of edestin, micro acetone titration (3) was used. Minced rabbit kidney was ex- tracted for 24 hours with 10 volumes of 60 per cent glycerol. The filtrate, diluted 1: 6 with water, was used for the enzymatic

421

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422 Enzymatic Histochemistry. III

determinations. To 7 c.mm. of kidney extract were added 7 c.mm. of water or of cysteine solution (10 mg. in 1 cc.) at the proper pH, 7 c.mm. of veronal-HCI buffer (4), and 7 c.mm. of 5 per cent clupein sulfate or 5 per cent edestin solution at the required pH. The rate of decomposition was measured after 4 hours incubation at 37”, as described in a previous paper (5). The results obtained have been presented in Table I. In the same table have been shown the results of studies on kidney

TABLE I

pH Optima of Kidney Cathepsin, Dipeptidase, and Aminopolypeptidase

The activity is expressed in c.mm. of 0.05 N HCI.

Substrate

Clupein sulfate

Clupein sulfate + cysteine

Edestin* “ +

cysteine

Glycylgly- tine

dl-leucyl- diglycine

-

1.0

.62

3.5

.6:

.Of

4.0

.a

.21

.3(

.4(

T 6.0 6.5 7.0 7.5 8.0 8.6

2.06 1.88 0.42

5.04 4.66 2.66

1.962.202.482.622.502.3:

1.562.883.003.203.062.61

Enzyme

Cathepsin

I‘

“ I‘

Dipepti- dase

Amino-

POlY-

pepti- dase

* Edestin precipitates at higher pH and therefore only the acid side could be investigated.

dipeptidase and aminopolypeptidase contained in the same extract. 7 c.mm. of 0.2 M glycylglycine or 7 c.mm. of 0.2 M

dl-leucyldiglycine were added to 7 c.mm. of kidney extract, and after 1 hour’s incubation at 25” the increase of amino groups was measured by micro acetone titration (3). The results are reported in c.mm. of 0.05 N HCI.

As indicated in Table I, the pH optimum for clupein sulfate decomposition was at 5, while the optimum for edestin was found

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L. Weil and R. K. Jennings 423

to be at pH 4. In both cases the addition of cysteine produced a marked activation, indicating that the enzyme responsible for the protein decomposition is of the catheptic type. Both the dipeptidase and the aminopolypeptidase of the kidney had pH optima at 7.5.

Determinations were also made of the decomposition rate of the above substrates under the action of the enzymes. By confining our experiments so that the values obtained were within the linear portion of the curve, these values could be used as a measure of the enzyme concentration.

FIG. 1. The rates of enzymatic decompositions of various substrates. X dipeptidase activities, 0 aminopolypeptidase activities, l esterase activities, A cathepsin activities, !I amylase activities.

The results obtained with kidney esterase and amylase have been included in Fig. 1. The following procedure was used in making these determinations. 7 c.mm. of a solution containing 2 per cent Lintner starch, 1 per cent NaCI, and 0.1 M phosphate buffer at pH 6.8 were added to 7 c.mm. of kidney extract. After each incubation period at 37” the increase of glucose was measured by the method of Linderstrom-Lang and Holter (6). For the kidney esterase determination, the method of Glick (7) was used. 7 c.mm. of kidney extract were employed, to which were added 7 c.mm. of methyl butyrate emulsion which had been prepared according to the method of Glick and then treated in the supersonic

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424 Enzymatic Histochemistry. III

oscillator to obtain a stable emulsion. Because of the high activity of kidney esterase the incubation temperature was low- ered to 25”.

The preparation of rabbit kidney for histoenzymatic investiga- tion was carried out in the same manner as that described in a previous paper (1). After saline perfusion, a tissue column was cut parallel to the flat side of the kidney; a borer of 2.5 mm. diam- eter was passed by means of a drill-press first through the pelvis, then the medulla, and finally the cortex. In this way a column was obtained containing all the structural elements of the kidney. The freezing-microtome and the histological technique were the same as were previously employed (8). Unfortunately, the freezing-microtome technique of Linderstrecm-Lang and Mor- gensen (9) could not be used in this work because of the damaging effect of freezing upon the cells of the kidney. The frozen tissue slices proved unsuitable for quantitative histological work.

For the histoenzymatic estimation of kidney cathepsin two adjacent slices 15 p in thickness were placed in a micro test-tube charged with 7 c.mm. of water. The catheptic activity of the slices was estimated after a 4 hour incubation period as described above, with edestin as the substrate. Only the full activity (with cysteine) was measured.

In the histoenzymatic estimation of dipeptidase and amino- polypeptidase only one 15 ,u slice was placed in the tube containing 7 c.mm. of water, and the determination carried out as described. Owing to the high activity of these two enzymes in the rabbit kidney it was necessary to reduce the incubation to 1 hour at 25”. For the estimation of esterase one 15 p slice was used under the conditions described for the peptidase. Kidney amylase deter- mination was made on a 20 p slice by the technique described above with the exception that an incubation period of 20 hours at 37” was employed.

Enzymatic and Cellular Distribution in Rabbit Kidney

In order to ascertain which types of cells in the rabbit kidney were responsible for the catheptic and aminopolypeptidase activities a correlation between the enzymatic activity and the number of various cells had to be demonstrated. Parallelism between the enzyme curve and the curve representing cellular

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L. Weil and R. K. Jennings 425

counts should serve as evidence of such relationship. The tech- nique of making cellular counts was that used by Linderstrom- Lang, Holter, and Ohlsen (8) and Weil and Ely (1).

I 1

- length 01 the tissue column in mm

FIG. 2. The distributions of cathepsin and aminopolypeptidase activities and of various cells in rabbit kidney. O--- cathepsin activities, X aminopolypeptidase activities, W cells of the proximal tubules, A cells of the distal tubules, 0. relative areas of glomeruli, O-a- total numbers of cells of proximal and distal tubules, 0 total number of cells, .A corrected total number of cells.

An effort was made to correlate the catheptic and aminopoly- peptidase activities with the number of individual cells of the rabbit kidney. Figs. 2 and 3 show that such a correlation was not obtained, although the curves obtained with cathepsin and aminopolypeptidase run parallel, indicating that the two enzymes

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426 Enzymatic Histochemistry. III

are similarly distributed throughout the organ. The fact that both enzymes could be detected along the entire length of the tissue column indicated that they were not associated with a single type of kidney cell alone, and that it was desirable therefore

Cortex Pelvis

3 4 5 6 Le tissue column in mm

FIG. 3. The distributions of cathepsin and aminopolypeptidase activi- ties and of various cells in the rabbit kidney. Op- cathepsin activi- ties, X aminopolypeptidase activities, q cells of the t.ubules of the de- scending Henle’s loop, O-e- total number of cells of tubules of the descending and ascending Henle’s loop, 0 cells of the collecting tubules, .A cells of tubules of the ascending Henle’s loop.

to take into account all possible variations of the cellular counts. The cortical part of the kidney, as Fig. 2 indicates, consists chiefly of the proximal and distal tubules, and since no relation appeared to exist between the enzyme activities and the number of cells of

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L. Weil and R. K. Jennings

either type alone, the possibility that both types of cells are responsible for the enzymatic activities in the renal cortex was considered. Graphic comparison of the enzymatic activities with the total number of cells from proximal and distal tubules combined (Fig. 2) strongly supports this possibility.

The medullary portion of the rabbit kidney, consisting chiefly of the cells of the ascending and descending tubules of Henle’s loop as indicated in Fig. 3, showed a marked catheptic and amino- polypeptidase activity, though less than that of the cortex. The enzymatic activities again compared graphically with the total cell count, this time of the ascending and descending tubules of Henle’s loop, showed a definite relationship.

In the pelvic portion of the kidney tissue column the histological structure is very simple, consisting, as indicated in Fig. 3, only of cells of the collecting tubules. Since catheptic and amino- polypeptidase activities were demonstrable in this portion, such enzymes must be present in cells of the collecting tubules although the concentration of these enzymes was much lower than in the cortex or medulla.

In view of both enzymatic activities in all structural ele- ments of the rabbit kidney, a parallel between the graphical rep- resentation of the total number of cells at given levels and the enzymatic activity at corresponding points might have been expected. The parallelism so obtained, however, is of a qualita- tive character in that the elevations and depressions of the en- zymatic curves parallel the elevations and depressions of the curve of the cell count only in the sense of being similarly located, while differing in height. Of course, entire parallelism could be ex- pected only if all the various cells were presumed to possess the same enzyme concentration. Dividing the enzymatic activity at a given level by the corresponding total number of cells pro- vided an easy means of estimating the enzyme concentration of a single cell at that point. When this was done, it was found that the cells of the cortical region possessed about twice the enzymatic activity of the medullary cells, and about 4 times that of the cells in the pelvis, which is to say the cells of the collecting tubules. Since the cortex consisted primarily of proximal and distal tubules, the medulla of ascending and descending tubules of Henle’s loop, and the pelvis of collecting tubules, it could be

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428 Enzymatic Histochemistry. III

assumed that one cell of a convoluted tubule (proximal or distal) was the enzymatic equivalent of two cells from Henle’s loop and of four cells from the collecting tubule. Having made this assumption, the graphical representation of the cell count was corrected as follows: the total number of proximal and distal convoluted tubule cells was recorded as found, the number of cells of Henle’s loop was divided by 2, and the number of cells of collecting tubules divided by 4. The curve of the corrected total number of cells not only retained the qualitative parallelism with the enzymatic curve, but showed in addition a definite quan- titative correlation. The results, therefore, appeared to justify the assumptions made concerning the relative enzymatic content of the various types of cells.

The conclusions drawn from this experiment were that the catheptic and aminopolypeptidase activities might be found in all the cells of the rabbit kidney, while the quantitative distribution varied with the type of cell. The cells of the convoluted tubules were capable of about twice the activity of the cells of the loop of Henle, in the case of cathepsin and aminopolypeptidase, and about 4 times that of the cells of the collecting tubules.

Owing to the high enzymatic activity of the cells of the proximal and distal convoluted tubules, the enzyme content of the glomeruli could not be estimated. To decide this question it would be necessary to isolate pure glomerular tissue.

A second experiment carried out in the same manner con- firmed this conclusion; the results have been presented in Fig. 4. For the sake of clarity Fig. 4 shows only the curve for cathepsin together with a curve representing the corrected total number of cells as in Fig. 2. The correlation so obtained again appeared to be satisfactory.

The location of the catheptic and aminopolypeptidase activities in the rabbit kidney having been established, the catheptic activity was used as a guide for the localization of dipeptidase, esterase, and amylase activities in the subsequent experiments. This method was less time-consuming than that involving the cell counts and proved to be very helpful. In order to localize each enzyme a simultaneous determination of catheptic activity was made at each chosen level in the tissue column. Fig. 5 indicates the histoenzymatic distribution of dipeptidase and

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L. Weil and R. K. Jennings 429

catheptic activities. The curves were found to be definitely parallel in the cortical and medullary regions, while the pelvis was practically free of dipeptidase activity under the experimental

cortex

Medulla Pelvis

3 4 5 6 7

-.

,

P

-----length ok the tissue column in mm

FIG. 4. The correlation of cathepsin activities and the corrected total number of cells in the rabbit kidney. 0 cathepsin activities, q corrected total number of cells.

-length 01 the tissue column inmm

FIG. 5. Histoenzymatic distributions of cathepsin and dipeptidase activi- ties in rabbit kidney. X cathepsin activities, 0 dipeptidase activities.

conditions used. It was concluded that the distribution of kidney dipeptidase was the same as that of cathepsin and aminopoly- peptidase in the cortical and medullary region of the rabbit

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430 Enzymatic Histochemistry. III

in FIG. 6. Histoenzymatic distributions of cathepsin and esterase rabbit kidney. 0 cathepsin activities, X esterase activities.

7 Pelvis Medulla

d I I

activities

’ -length 4 the tissue column in mm

FIG. 7. Histoeneymatic distributions of cathepsin and amylase activities in rabbit kidney. X cathepsin activities, 0 amylase activities.

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L. Weil and R. K. Jennings 431

kidney, but was absent, or present only in very low concentration, in the pelvis and therefore in the cells of the collecting tubules.

In Fig. 6 the activities of kidney esterase have been compared with the catheptic activities in the same way that the dipeptidase was compared in the previous experiments. A marked parallelism throughout the entire length of the tissue column strongly sup- ported the conclusion that the histoenzymatic distribution of kidney esterase was the same as that indicated for cathepsin and aminopolypeptidase in Figs. 2 and 3.

Experiments carried out in the same way on kidney amylase led to a different picture, as shown by Fig. 7. The amylase ac- tivities followed closely the catheptic activities in the cortical region of the rabbit kidney. As soon as the medullary portion of the kidney was reached, the amylase activity disappeared, reappearing again in the pelvis where it again paralleled the catheptic activities. The conclusion appeared justified that the source of amylase activity in the cortex was the same as that of catheptic activity, while the cells of Henle’s loop (constituting the bulk of the medullary portion of the kidney) were practically free from amylase activity’ although possessing considerable catheptic activity. In the pelvic region, and therefore in the collecting tubules, the catheptic and amylase activities were again parallel.

SUMMARY

Histoenzymatic investigations have shown that catheptic, aminopolypeptidase, and esterase activities may be demonstrated in all structural elements of the rabbit kidney. The quantitative distribution of these enzymes, however, varied among the dif- ferent types of kidney cells; that is, the cells of the proximal and distal convoluted tubules were about twice as active enzymatically as the cells of the ascending and descending tubules of Henle’s loop and about 4 times as active as cells of the collecting tubules.

i It should be mentioned that some of the cells of the collecting tubules were present in the medullary portion of the kidney (see Fig. 3), and it was therefore surprising that their presence did not demonstrably influence the amylase activity in this region, since the cells of these tubules did exhibit this activity in the pelvic region although to a lesser degree than do the cells of the cortex.

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432 Enzymatic Histochemistry. III

The same distribution was found for dipeptidase activities with the exception that cells of the collecting tubules did not possess any of this activity under the conditions of the experiment. Amylase activity in the rabbit kidney was found to be associated with the ceils of the proximal and distal convoluted tubules and with the cells of the collecting tubules. The cells of the loops of Henle were free from amylase activity.

BIBLIOGRAPHY

1. Weil, L., and Ely, J. O., J. Biol. Chem., 112, 565 (1935-36). 2. Weil, L., Biochem. J., 30, 5 (1936). 3. Linderstr@m-Lang, K., and Halter, H., in Nord, F. F., and Weiden-

hagen, R., Ergebnisse der Enzymforschung, Leipzig, 3, 315 (1934). 4. Michaelis, L., J. Biol. Chem., 87, 33 (1930). 5. Weil, L., and Russell, M. A., J. Biol. Chem., 126, 245 (1938). 6. Linderstdm-Lang, K., and Holter, H., Compt.-rend. trav. Lab. Carls-

berg, SBrie chim., 19, No. 14 (1933). 7. Glick, D., 2. physiol. Chem., 223, 252 (1934). 8. Linderstdm-Lang, K., Holter, H., and Ohlsen, A. S., Z. physiol. Chem.,

227, 1 (1934). 9. Linderstrplm-Lang, K., and Morgensen, K. R., Compt.-rend. trav. Lab.

Curlsberg, Serie chim., 23, No. 4 (1938). by guest on October 25, 2020

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Leopold Weil and Robert K. JenningsOF ENZYMES IN RABBIT KIDNEY

DISTRIBUTIONHISTOCHEMISTRY: III. INVESTIGATIONS IN ENZYMATIC

1941, 139:421-432.J. Biol. Chem. 

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