Nephritis; an experimental and critical study of its nature, cause and the principles of its relief

Front matter

THE LIBRARY

OF

THE UNIVERSITY

Preface

The following pages constitute a continuation of the experimental studies in clinical physiology which were published last year under the title (Edema} The second half of the present volume brings a detailed account of observations touched upon only incidentally there, which show how some of the severest grades of nephritis which are generally held to present considerable difficulties in treatment may be easily relieved. The methods adopted to accomplish this purpose are in no small way different from certain forms of practice common to-day. Any hope that they may find general acceptance can therefore be entertained only if the principles upon which the suggested therapy is based have been correctly enunciated. What to my mind these are constitutes the first half of the volume.

It is impossible to mention by name all those of my friends and colleagues who with suggestion and criticism have helped along the days' work. Mr. Thomas Kelly and Dr. William H. Strietmann have been enthusiastic and willing coworkers; Prof. Lauder W. Jones helped me with his store of chemical knowledge; Mr. Alfred Brodbeck took interest in the observations made on athletes; Dr. Charles Goosman gave of his time and skill to prepare the photomicrographs ; Mr. Charles Hecker and Mr. Peter Scherrer of theirs to make the remaining photographs; Mr. Paul M. Stewart arduously deciphered the manuscript. Very sincere thanks are tendered all of them.

Martin H. Fischer.

University of Cincinnati, June, 19 II.

^ CEdema. A Study of the Physiology and the Pathology of Water Absorption by the Living Organism. New York, 19 10. John Wiley and Sons, Publishers.

vii

Table Of Contents

Introductory Paragraph.

I. The Albuminuria: Page

1. Introduction 2

I. H2o

The appearance of the fibrin in each of .the flasks at the end of this time is shown in Fig. i. In the first three flasks (i, 2, 3) there is a progressive increase in the swelling of the fibrin with the progressive increase in the concentration of the acid. Beyond this point (flasks

Fig. 2.

4 and 5) there is a decrease in the swelling in spite of the further increase in the concentration of the aci^. The least amount of swelling is noted in flask 6 which contains water only. The solution of the fibrin is indicated in Fig. 2. From left to right these tubes correspond with the flasks of Fig. i. No precipitate of albumin is seen in the tube on the extreme right, indicating that the fibrin did not go into solution in the water (neutral reaction). All the remaining tubes show a precipitate of albumin.

Experiment 2. — 0.5 gram of powdered fibrin is put into each of the following solutions and shaken for 5 hours :

1. 10 c.c. To normal HCl + 40 c.c. H2O.

2. 10 c.c. TO- normal HCl + 40 c.c. \ molecular NaCl.

3. 10 c.c. tV normal HCl + 40 c.c. \ molecular NaCl.

4. 10 c.c. To normal HCl + 40 c.c. \ molecular NaCl.

The amount of albumin that went into solution is indicated in Fig. 3. No precipitate is seen in the tube on the extreme right

Fig. 3.

(water). Most albumin is found in the first tube (pure acid solution;. It is evident that the presence of the sodium chloride reduces the amount of the albumin that goes into solution. The amount of this reduction is the greater the higher the concentration of the salt.

Experiment 3. — 0.5 gram of powdered fibrin was placed in each of four flasks containing the following solutions and shaken for 5 hours:

XEPHRITIS

1. lo c.c. i\t normal HCl + 40 c.c. HjO.

2. 10 c.c. 15 normal HCl + 40 c.c. | molecular Xa2S04.

3. 10 c.c. Jo normal HCl -i- 40 c.c. | molecular MgSO*.

4. 10 c.c. A normal HCl -|- 40 c.c. | molecular CuSO^. After filtering, the amount of albumin dissolved in the supernatant

liquid found above the fibrin in each of the flasks was determined by mixing 20 c.c. of filtrate with 14 c.c. phosphotungstic acid. The re-

FiG. 4. suit is shown in Fig. 4. As is readily apparent, each of the salts markedly reduced the amount of albumin that was dissolved.

Experiment 4. — 0.5 gram of powdered fibrin was introduced into each of five flasks containing the following solutions and shaken for 5 hours:

1. 10 c.c. ^6 normal HCl -f- 40 c.c. | molecular sodium acetate.

2. 10 c.c. 15 normal HCl -j- 40 c.c. i molecular sodium nitrate.

3. 10 c.c. tV normal HCl + 40 c.c. | molecular sodium sulphate.

4. 10 c.c. ^6 normal HCl + 40 c.c. i molecular sodium citrate.

The relative amounts of albumin found dissolved in each of these mixtures at the end of this time are indicated in Fig. 5. As is again

Fig. 5. evident, most albumin was dissolved by the pure acid solution. Each of the salts decreased through its presence the amount thus

dissolved.

{h) Gelatine. — What has been said under paragraph (a) regarding tlte "solution'' of fibrin holds almost word for word for the " solution " of gelatine. The best commercial gelatine shows some solubility in water. The commercial product, as is well known, has a decidedly acid reaction. WTien, instead of being placed in water, gelatine is dropped into solutions of acids (or alkalies) this solubiHty of the (commercial) gelatine is greatly increased. The presence of neutral salts in the acid (or alkah) solution decreases the amount of the gelatine that will go into solution. As in the case of fibrin we note here again a progressive decrease in the amount that " dissolves " with every increase in the concentration of the added salt. With a given concentration, the amount of such a decrease varies with the salt employed, and here again it seems that monovalent ions do not as a group decrease the " solubility " of the gelatine as much as bivalent ions, or these as much as trivalent

ions.

The following experiments will serve in illustration of what has been said.

Experiment 5. —

- The fol

lowing solutions we

I.

o.ooi normal HCl.

o.oi normal HCl.

Five leaves of dry gelatine, each measuring 3I by i^ cm., weighing altogether 0.5 gram, and obtained by cutting them out of the central portions of the large gelatine leaves that are obtained commercially, were dropped into each of these solutions. From time to time the dishes containing the solutions with their gelatine leaves were agitated so as to keep the gelatine from adhering to the sides, and aid the solution of the gelatine. All the vessels were treated exactly alike. The degree of solution of the gelatine after 28 hours in these various solutions is indicated in Fig. 6. As the photograph shows, least gelatine is dissolved in the pure water. With the increase in the concentration of the acid there is a progressive increase in the amount of dissolved gelatine, but only up to a certain point, after which it falls in spite of the continued further increase in the concentration of the acid.

Experiment 6. — In the manner just described, 5 leaves of dry gelatine, weighing in toto 0.5 gram, and of the same surface were placed in each of the following solutions:

100 c.c. HoO. 15 c.c. To normal HCl + 85 c.c. H2O.

15 c.c. tV normal HCl + 2| c.c. f mol. NaCl + 82^ c.c. H2O. 15 c.c. tV normal HCl + 5 c.c. f mol. NaCl + 80 c.c. H2O. tV normal HCl + 10 c.c. i mol. NaCl + 75 c.c. HoO.

15 c.c. tV normal HCl + 15 c.c. f mol. NaCl + c.c

H2O.

■T

]Hff

rl^^^l

^Hj mm

■hj

Q

Fig. 6.

The relative degree of solution of the gelatine after a residence in these mixtures of 18 hours is indicated in Fig. 7. The addition of the salt has decreased the amount of the gelatine that goes into solution in the hydrochloric acid, and this the more the higher the concentration of the added salt.

Experiment 7. — Five leaves of dr>' gelatine weighing altogether 0.4 gram and having the same surface were placed in each of the following solutions:

1. iscciVn. HCl +85 c.c. HoO.

2. 15 c.c. \Q n. HCl + 10 c.c. \ mol. sodium acetate -f- 75 c.c. HoO.

3. 15 c.c. T(y n. HCl -f 10 c.c. \ mol. sodium chloride + 75 c.c. HoO.

4. 15 c.c. tV n. HCl -f 10 c.c. \ mol. sodium nitrate + 75 c.c. HoO.

5. 15 c.c. tV n. HCl -f 40 c.c. \ mol. disodium hydrogen phosphate

.

Fig. 7.

1 1 1' 11 '

l«llll«

Fig.

6. 15 c.c. tV n. HCl + 40 c.c. \ mol. sodium sulphate + 45 c.c. H2O. 7.15 c.c. tV n. HCl + 10 c.c. T mol. sodium citrate + 75 c.c. H2O.

The relative amounts of gelatine dissolved in these various solutions after the gelatine leaves had with occasional agitation remained in them for 19^ hours are indicated in Fig. 8. As is readily evident, each of the salts decreases by its presence the amount of gelatine dissolved in the acid solution. The divalent and trivalent anions are more powerful in this respect than the monovalent ions, with the exception of the acetate. The intermediate position taken by this ion, in this matter of the solution of the gelatine, corresponds with the intermediate position occupied by this same ion in the swelling of the colloid under similar circumstances.

It is clearly apparent from what has been said that the " solution " of two typical emulsion colloids (protein gels) is intimately connected with the character of the medium in which these gels find themselves. Acids (and alkalies) favor their solution while various other substances (notably salts) either do not affect the solubility of the gel at all, or if present in conjunction with an acid (or alkali) depress the amount that would have been '' dissolved " if the acid (or alkali) had been present alone.

We will now adduce the evidence which is intended to show that the colloidal gel that constitutes the urinary membrane goes into ^^ solution " in the urine {albuminuria results) under the same conditions under which fibrin or gelatine gels go into " solution " in water.

5. Albuminuria as a Phenomenon Identical with the " Solution " of a Protein Gel.

No special argument is necessary to prove that what lies between the urine on the one hand and the blood on the other {the kidney) represents physicochemically a colloidal gel. The general physical properties of the kidney as a whole betray this fact, and from chemical analysis we know that the albumins, globulins, higher carbohydrates, fats, and

" lipoids " which constitute, exclusive of water, the bulk of this organ, are all to be counted in the group of our most tj-pical emulsion colloids. What we need to show is that under normal circumstances, in " health," the conditions are such as to keep these colloids (as we are discussing albuminuria this means the proteins) in the gel state, while under other circumstances (for example in nephritis) conditions are offered which permit these colloids to pass over into the sol state and so escape with the urine. As we found changes in the reaction of the medium to play a most important role in the '^ solution " of such emulsion colloid gels (fibrin and gelatine), we will direct our chief inquiry into the question of whether such changes occur in the kidney in every condition characterized by an albuminuria. An ahnormal production or accumulation of acid ijt the kidney lies at the bottom of every albuminuria and is responsible for it. Our support for such a \dew will come from three directions.

1. Evidence of an abnormal production or accumulation of acid in the kidney, or conditions predisposing thereto, exist in every case of albuminuria, and conversely;

2. Any means which leads to an increased production or favors the accumulation of acid in the kidney results in albuminuria.

3. Any means by which we can decrease the '' solubility " of various protein gels (fibrin and gelatine) in an acid medium constitutes a means by which we can decrease albuminuria.

The first two of these we will consider at once. The third we will take up separately later in this paper when we discuss the subject of the treatment of nephritis.

I. If albuminuria is to be regarded as a process of " solution " of the urinary membrane in consequence of the presence of acids in it, then evidently the maintenance of the gel state of the normal membrane must be intimately associated with a maintenance of neutrality in it. We are therefore first of all interested in the fact that (exclusive of the gastric juice, the urine, and less positively, the sweat, vaginal secretion, and alimentary contents when fat is fed) the fluids and tissues composing the living mammal are to all intents and purposes neutral in reaction and are capable of maintaining this neutrality against the introduction of considerable acid into them.

In the terms of our modern physical chemistry, an acid reaction is due to the presence of free hydrogen ions, an alkaline reaction to the presence of free hydroxyl ions. A neutral reaction means, therefore, one of two things: either neither of these ions are present, or else just as many of the one as of the other, so that they balance each other (the latter is the case in the body). The neutral reaction of the blood (and from this it has been generally assumed that the tissues themselves are also neutral in reaction) seems at first sight a rather surprising fact when considered in the light of our older teachings that the body fluids and the cells are all " alkaline." But these older teachings are erroneous for they are based upon an improper interpretation of the results obtained with titration methods. The blood, for example, has generally been held to be " alkaline " because it is capable of neutralizing acid. But as we know now, the power of a solution to neutralize an acid is no index of its content of free, that is to say, active, hydroxyl ions which is the true measure of its alkalinity. For a proper measure of this hydroxyl ion concentration in the blood (or in the tissues) all the determinations are valueless that antedate the fundamental measurements of P. Fraenkel,^ G. Farkas,^ and Rudolf Hober ^ who first used proper physicochemical methods (so-called gas chains) in the biological study of this problem. The observations of all these authors agree in pronouncing the normal blood neutral in reaction ; as neutral as pure distilled water.

Of further interest is the fact that this state of neutrality of the blood {and of the tissues) is maintained against the introduction of considerable acid or alkali into them. When exposed to the action of an acid, it is found that the normal hydrox>'l ion concentration of the blood drops with the progressive introduction of acid into it in the form of a curve, which falls only very slowly at first, and then more rapidly. Just why and how the state of neutrality is thus maintained for a period does not at this particular moment interest us, but it may not be amiss to point out that two factors are involved in the process. The first lies in the fact that such salts as sodium carbonate and disodium hydrogen phosphate are capable of uniting with acids (carbonic and phosphoric acids) to form salts having a higher hydrogen content (sodium bicarbonate and sodium dihydrogen phosphate), but which in their dissociation yield few more hydrogen ions than the salts from which they were originally formed and which were present in the blood to start with. In other words, there is for a time only a slight increase in the concentration of the hydrogen ions (increase in acidity) in spite of the considerable introduction of free acid into the system. (Z. /. Eenderson.y

^ P. Fraenkel: Pfluger's Archiv, 96, 6oi (1903).

2G. Parkas: Pfluger's Archiv, 98, 551 (1903); Archiv f. (Anat. und) Physiol., Supplement, 517 (1903).

*L. J. Henderson: American Journal of Physiology, 16, 257 (1906); 21, 169 (1908); 21, 427 (1908); Ergebnisse d. Physiologic, 8, 257 (1909), where extensive references to the literature will be found-

The other and lesser element for the maintenance of neutrality resides in the amphoteric character (that is to say their power of combining either with acids or alkalies) of the colloids found in the blood or the tissues. The albumins, for example, can unite with considerable quantities of acid (or alkali) without any decided change in their behavior toward indicators. The presence of certain colloids in any system will therefore serve to delay the increase in the concentration of the hydrogen ions when an acid is added to this system.^ But let not the impression be gained from these remarks that the blood or the tissues are not sensitive to even very minute additions of acid (or alkali) to them. Such an increase in the concentration of the carbonic acid as occurs when normal arterial blood becomes venous is already sufficient to reduce the hydroxy! ion concentration in the latter to one half that existing in normal arterial blood. How profoundly even such a change affects the state of the colloids we will have occasion to discuss later. For the present we are content with making the point that the blood and (presumably) the tissues are neutral in reaction, that they are capable of maintaining this neutrality within rather wide limits, even when subjected to the action of an acid, and that in consequence, under normal circumstances, conditions are such in the cells of the kidney as to inaintain the colloids here in their gel state.

2. As modern physicochemical studies have reduced what we formerly regarded as the " alkalinity " of the blood to a point where we may call it neutral, so also have they reduced the normal " acidity " of the urine from what we used to assume this to be. Just as the neutralizing power

^ J. Sjoqiiist: Skand. Arch. f. Physiol., 5, 277 (1895); Otto Cohnheim: Zeitschr. f. Biol., 33, 489 (1896); K. Spiro and W. Pemsel: Zeitschr. f. Physiol. Chem., 26, 233 (1898), 5. Bugarszky and L. Liehermann: Pfliiger's Arch., 72, 51 (1898); r. B. Robertson: Jour, of Physical Chem., 11, 542 (1907); 12, 473 (1908).

of the blood for acids is no indication of its true reaction, so also is the amount of alkali with which a given specimen of urine will combine no measure of its true, that is to say, active, acidity. To gauge this properly the concentration of the hydrogen ions in it must be determined and this was not done until von Rhorer ^ and Rudolf E'dher ^ applied the principle of the gas chain to the physicochemical analysis of the urine. The following Table I, taken from Hoher,^ indicates what is the concentration of the hydrogen ions in a series of normal morning urines. This gives a measure of their true acidity.

TABLE I. — NORMAL URINE.

Hydrogen ion acidity (IO-5 . Ch).

Titration acidity.

It would support our idea of the cause of albuminuria if it could be shown that this acidity of the urine increases in conditions associated with an albuminuria. How strikingly true this is, is clearly evident from the following analyses of nephritic urines also taken from E'dher and made of course without thought of using them for such purposes as we do here.

As is clearly evident on comparing these two tables the active acidity of the urine of nephritics may be more than four times that of the normal urine. But Table II already suffices to betray another fact. The highest acidities occur

1 L. von Rhorer: Pfliiger's Archiv, 86, 586 (1901),

2 Rudolf Ilobcr: Hofmeister's Beitrage, 3, 525 (1903).

3 Rudolf Ilobcr: Physikalische Chemie d. Zelle u. d. Gewebe. Zweite Aufl. 158. Leipzig, 1906.

in the acute jorms of nephritis, in other words, in the same forms in which we find most albumin. The lowest values are found in the chronic interstitial forms, in other words, in the very types in which albumin is very likely to be found only in traces or at times not at all. The degree of the albuminuria therefore follows very closely the degree of acidity. We shall have occasion to return to this question later.

TABLE 11. — ABNORMAL URINE.

Hydrogen ion

acidity

Titration acidity.

Remarks.

0.019"]

Interstitial nephritis.

Acute nephritis.

Chronic interstitial nephritis.

Let us now look at the columns in these tables that record the titration acidities. It is such values — erroneously interpreted as measures of the true acidity of the urine — that we find recorded in all the studies of nephritis. When the individual titration acidities in the above tables are compared with their corresponding active acidities as determined by measuring the hydrogen ion concentrations in the urine, it is readily apparent that the two values do not even approximately parallel each other. What is learned when the titration acidity of the urine is determined is its capacity to neutralize alkali. Under otherwise constant conditions it is clear that this titration acidity of the urine must grow with every increase in the amount of acid in the urine. The uniformly higher titration acidity of the urine in nephritis, as is shown not only in the above tables I and II but by the scores that may be found in any of the larger works that deal with this question of nephritis, becomes further evidence therefore in favor of our contention that an abnormal production or an abnormal accumulation of acid occurs in the kidney in every albuminuria.

3. In the same way that we use the increased alkah capacity of the urine as e\idence for the presence of abnormally large amounts of acid in it (and so in the kidney cells from which this comes), so also may w^e use the decreased acid capacity of the blood as evidence in the same direction. The titration values of the blood, w^hich the older clinical observers looked upon as indices of its " alkalinity," may be drawn upon for evidence to show that in the albuminurias there exists a decreased power of the blood to neutralize acids. As studied particularly by Rudolf von Jaksch,^ W. H. Rionpf,^ E. Peiper ^ and F. Kraus ^ a decrease in the acid capacity of the blood is noted in no conditions more strikingly than in nephritis and its oft associated urcEmia.

4. Our argument thus far has shown that in nephritis there is a great increase in the true acidity of the urine, and that in both the urine and the blood there occur changes in the titration values which clearly indicate that both are holding a more than normal amount of acid. Our knowledge of physical chemistry (the laws of chemical equilibrium) permits us to utilize these facts as e\adence indicating that the tissues of the kidney which He between the urine on the one hand and the blood on the other (all that we in our definition sum up as the urinar^^ membrane) must, under such circumstances, also show a rise in acid concentration. But it would further strengthen this view if we could bring a more direct proof in support of this de-

2 W. n. Rumpf: Centralbl. f. klin. Medicin, 12, 441 (1881). ȣ. Peiper: Virchow's Arch., 116, 337 (1889).

*F. Kraus: Zeitschr. f. Heilkunde, 10, 106 (1889); Archivf. exp. Path. u. Pharm., 26, 181 (1889).

duction. It would be well of course if wx could obtain a direct measure of the hydrogen ion concentration in the kidney. Gas-chain methods are naturally not applicable to solid organs, and to apply them to the expressed juice of the kidney would be to introduce so many errors into the whole problem as to render the conclusions valueless. We can, however, obtain material help by using indicators.

Proof of an increase in the amount of acid held by the kidney cells in conditions associated with an albuminuria is furnished by the following facts :

In 1885, E. Dreser ^ described a series of experiments on the excretion of dyes by the kidney which differed from the preceding studies of this subject as first made by R. Heidenhain ^ and M. Nussbaum,^ in that he utilized the results of his experiments in an attempt to get an answer to the question as to where in the kidney the acid of the urine is secreted. Dreser made chief use of acid fuchsin which he injected in 5 to 10 per cent solutions (amounts not stated) into the dorsal lymph sacs of frogs. This dye has the property of being red in aqueous solution only in the presence of an acid; in an alkaline solution it becomes practically colorless (yellow). Dreser therefore reasoned that the presence of a red color in any tissue after the injection of this dye into the circulation of an animal was evidence for an acid reaction in that tissue. The first fact noted by Dreser that is of interest to us is that after a single dose of acid fuchsin the urine is found shortly thereafter to become brilHantly red. If the kidney from such an animal is examined no stained cells are noted anywhere in the kidney. To interpret this fact we would have to say that normally the urine is acid in reaction hut the cells of tJie

2 R. Heidenhain: Pfliiger's Archiv, 9, i (1875).

^ M. Nussbaum: Pfliiger's Archiv, 16, 141 (1878).

normal kidney have not an acid reaction. The following may serve to corroborate this finding of Dreser.

Experiment 8. — Three frogs, weighing 35 grams each, are injected, respectively, with 0.25, 0.5, and i.o c.c. of an aqueous i per cent acid fuchsin solution, into the dorsal lymph sac. All are seen to secrete a red colored urine before being killed. They are killed respectively after i, i^ and 4^ hours. On autopsy, red urine is found in the bladder of each anitnal. The kidneys are not stained. They are rapidly removed from the freshly killed animals, frozen with liquid carbon dioxide gas (on a Bardeen freezing microtome where the gas does not come in contact with the tissue) and sectioned. The sections are immediately transferred to a slide (without being brought in contact with water or any other medium except air), covered with a cover slip, and examined under the microscope. None of the kidney tissues are seen to he stained. To be sure that the freezing plays no part in the findings, a parallel series of free-hand sections, and crush preparations of the kidneys are made. No stained cells are found.

When the uncolored sections are touched with very dilute acetic acid they are seen gradually to assume a pink color. Acid fuchsin is therefore present in the kidney tissues, but as cut from the body the reaction of this organ is not such as to allow its red color to appear. The pink tinge visible in the kidney after being touched with acid includes the glomeruli.

ExPERniEXT 9. — To show that what was said for the frog holds also for the mammal, two young rabbits, weighing, respectively, 184 and 189 grams, received into the ear veins 2.0 and 4.0 c.c, respectively, of a I per cent aqueous acid fuchsin solution. At the end of 30 and 35 minutes, respectively, they were killed by a blow on the head and immediately autopsied. Light red urine was found in the bladder of the first, deep red urine in that of the second. The appearance of the kidneys in both animals was entirely normal, and no dye was visible in the kidneys either macroscopically or microscopically. When a little very dilute acetic acid was permitted to flow under the cover slips, the sections" turned uniformly pink.

Drcscr noted no staining of the frog's kidney until he had repeated his acid fuchsin injections several times. Then he found that the cells of the convoluted and of the straight tubules began to stain red. He interpreted this finding by saying that from the long-continued effort on the part of these cells to excrete the dye, they become fatigued and so some of the dye remains behind to be discovered on subsequent section of the kidney. From all these facts Dreser concluded that the acid constituents of the normal urine are " secreted " by the convoluted tubules, and that since the glomeruli and their capsules remain unstained, the '' urine " coming from these must be '* alkaline " in reaction, to change to an acid reaction after passing by the convoluted tubules. Whether such conclusions are really justified we shall have occasion to discuss later.

No one can quarrel with the simple experimental finding that acid fuchsin does not stain the normal kidney, and does do this after repeated and long-continued injections. Such staining of the kidney Dreser still regards as '^ physiological." Strictly speaking, and for reasons that will be apparent as we go on, I should myself be more inclined to regard it as "pathological." What Dreser calls the ^^ fatigue " of the cells of those portions of the kidney which stain after repeated infections of the acid fuchsin, we are perfectly safe in regarding as the first evidences of an abnormal acid content in these cells, and we may hold that the repeated infection of this dye is itself responsible for such a condition.

Acid fuchsin is a weak acid, and must produce the same effects upon the kidney that we know are produced by the injection of any other acid.^ After the injection of acids we note regularly all the signs of a nephritis, and that these were not absent in Dreser's experiments is clearly evidenced by the " anuria " which this author so often noted in his frogs.