Kidney International, Vol. 16 (1979), pp. 404-408

Renal reabsorption and excretion of horseradish peroxidase1 WERNER STRAUS I)epartment of Biochemistry, University of Health Sciences, The Chicago Medical School, Chicago, Illinois

Horseradish peroxidase (HRP) was introduced as a tracer protein in 1957 to study the role of "hyaline droplets" in the reabsorption of proteins by kidney cells [1]. The work on hyaline droplets was inspired by Jean Oliver who had made a detailed histologic investigation of these cell granules. Since the begin-

that they were lysosomes [11, 15]. This conclusion was greatly facilitated by the isolation of cell granules with similar enzymic properties from liver cells

ning of the century, it had been a matter of controversy among pathologists as to whether the

droplet fractions was time-consuming and was only semiquantitative. When HRP was tried instead of egg white [11, the advantages of this tracer protein

by DeDuve et al [16]. The term "lysosome" was also introduced by these investigators. The serologic test for egg white in the isolated-

"hyaline droplets" of kidney cells were signs of secretion, absorption, or of cellular degeneration [21.

became apparent immediately. As an enzyme, it

Oliver concluded from his observations that the "droplets" function in the reabsorption and disposal of proteins [3-7]. Similar granules had been

could be measured readily in cell fractions by sensitive biochemical methods, and its localization in tis-

shown previously by Gerard and Cordier [8] and by

methods. It could be seen, for example, that a few minutes after i.v. injection, HRP appeared in pinocytic vesicles (phagosomes) in the apical cytoplasm of the proximal tubule cells (PTC) [17, 18]. The fusion of phagosomes with lysosomes could be observed by staining the lysosomes red by an azo-dye procedure for acid phosphatase and by staining, in the same tissue section, the phagosomes blue by the benzidine reaction for ingested HRP. Early after injection of HRP, the blue-stained phagosomes and

sue sections could be detected by cytochemical

Lambert [9] to be involved in the reabsorption of proteins in kidney cells of amphibians. Even earlier, von Möllendorff [10] had studied the storage ("ath-

rocytosis," "Speicherung") of colloidally dispersed dyes by granules of a similar type in kidney cells.

On the suggestion of Oliver, I isolated droplets from kidneys of rats after injection of egg white [11]. In collaboration with Oliver, it could be shown that the injected egg white was several times more concentrated in the isolated-droplet fraction than it

the red-stained lysosomes were separate. Later, most of the stained granules in the PTC's appeared purple, thus indicating the fusion of phagosomes and lysosomes [17, 19]. The gradual disappearance of HRP from the phagolysosomes, probably after catheptic digestion, could be followed in tissue sections and in cell fractions [17, 20]. Although most of the peroxidase in the kidney cells was concentrated in the phagolysosomes of the PTC's, HRP also appeared in phagolysosomes of the distal and collecting tubule cells [18-201. The changes occurring during the reabsorption of HRP in the PTC were stud-

was in the original homogenate [12], thus confirming the hypothesis of Oliver. The contributions of Oliver to the clarification of this problem were also described by Kretchmer and Bernstein [13]. Oliver believed that the "droplets" were derived

from mitochondria [3-7]. This opinion is understandable, for lysosomes had not yet been discovered. The existence of autophagic vacuoles (a type of lysosome) containing mitochondria [14] may also

have complicated the problem. The enzymatic properties of the isolated droplet fractions showed

ied at the ultrastructural level by Graham and

Received for publication March 21,

This paper is dedicated to the memory of Dr. Jean Redman Oliver, Distinguished Service Professor of Pathology, emeritus, State University of New York Downstate Medical Center. Dr. Oliver died on November 19, 1976.

1979

0085-2538/79/0016-0404 $01.00

© 1979 by the International Society of Nephrology 404

Straus

405

Karnov sky [211. Similar ultrastructural changes as

urine [20]. Preinjection of egg white prior to HRP

those occurring during the reabsorption of HRP, were observed during the uptake of hemoglobin

caused an 80 to 90% decrease of HRP uptake by the

[22—25], ferritin [26-281, and of homologous serum albumin [29—31]. These investigations are discussed in the excellent review by Maunsbach [32].

Because HRP has a relatively low molecular weight (40,000 daltons) [331, appreciable amounts of

it pass the glomerulus. In addition to HRP, other hemoproteins were used to study the permeability of the glomerulus, or that of other capillaries, in relation to molecular weights or to study renal protein uptake or excretion; catalase (mol wt, 240,000 daltons) [34, 35]; myeloperoxidase (mol wt, 160,000 daltons) [36]; lactoperoxidase (mol wt, 82,000 daltons) [37]; hemoglobin (mol wt, 68,000 daltons) [2225, 38-42]; myeloglobin (mol wt, 17,000 daltons)

[43, 44]; cytochrome c (mol wt, 12,000 daltons) [45], and "microperoxidase" (mol wt, 1,900 daltons) [46, 47]. It should be noted that among these hemoproteins only myeloperoxidase and lactoperoxidase show high peroxidatic activities comparable to that of HRP. Recently, Rennke, Patel,

and Venkatachalam introduced acid and basic

renal cortex, and the urinary excretion of HRP increased greatly [511. The observations suggested that the pinocytic uptake of HRP by the kidney cells had been saturated. A few comments may be made at this point on the

disappearance rates of HRP from the blood. After i.v. injection, the plasma concentration of HRP decreased in a logarithmic fashion, with a half-disappearance rate of approximately 30 mm [20, 49]. This observation was confirmed in systematic studies by

Jacques [50], who, in addition, showed that the plasma disappearance rates of HRP during the first 1 to 2 hours after injection decreased with increas-

ing dose levels. Afterwards, the disappearance rates of HRP increased again to approach the same rates for all dose levels. It may be suggested that the influence of the dose on the plasma disappearance rates of HRP is related to the temporary decrease of the urinary excretion of HRP occurring during the first 20 to 30 mm after the injection of a relatively

high dose of the protein as an effect of vascular leakage (see below). These effects are dose-dependent and do not occur at low-dose levels. Rodman, Schlesinger, and Stahl [52] recently showed that the plasma disappearance rate of HRP is greatly influenced by the injection of other glycoproteins. They

groups into HRP [48]. These modified HRP molecules are of great interest because they make it possible to investigate the influence of electric charge on membrane binding, absorption, and permeability for proteins.

concluded that the endocytic uptake of HRP is

Before discussing further the use of HRP as a tracer for the study of renal protein absorption and

mediated by specific sugar recognition sites on the cell membranes.

excretion, the fate of HRP in the whole body should

Quantitative biochemical studies. For quan-

be considered briefly. Appreciable amounts of injected HRP are also taken up by the liver and by the reticuloendothelial system [49]. Approximately 30% of HRP are excreted in the urine during the first 4 hours, and 6% of HRP are excreted in the feces from 6 to 20 hours after injection. Although the kidney takes up only 3 to 7% of injected HRP (increasing with age), the concentration of HRP in the kidney cortex is higher than it is in any other

titative work, the concentration of HRP in total particulate fractions of the renal cortex was taken as an approximate measure for renal proximal reabsorp-

organ [20, 491. Because of its size, the liver takes up more HRP than the kidney. The uptake by the liver tends towards saturation at relatively low-dose lev-

els, whereas the uptake by the kidney increases in proportion to the dose, up to high-dose levels [20, 50]. It is difficult to determine at what dose saturation of the kidney cells with HRP occurs because the animals become sick at high-dose levels. When a second relatively high dose of HRP was injected 4 hours after the first injection, little further uptake by

the kidney cortex occurred, and approximately double the amount of HRP was excreted in the

tion. These fractions contain all the phagosomes and phagolysosomes of the proximal tubule cells. It

is the opinion of most investigators that absorbed proteins do not reach the soluble cytoplasm or other cellular organelles of the proximal tubule cells besides the phagosomes and phagolysosomes [32, 53,

54]. After a single i.v. injection of increasing

amounts of HRP, the concentration of HRP in the total particulate fractions increased in proportion to the injected dose. After injection of a constant dose, the concentration of HRP in the total particulate fractions did not change much during the first 1 to 3 hours as long as an excess of HRP was present in the blood and the glomerular filtrate [20]. It was suggested that a constant fraction of HRP was absorbed from the glomerular filtrate into the proximal tubule cells by pinocytosis and that, after the fusion of phagosomes with lysosomes, the amount of HRP

406

Renal reabsorption and excretion of HRP

broken down in the phagolysosomes was replaced by newly ingested HRP [20]. The constancy of fractional uptake, and the apparent balance between ingestion and breakdown, made it possible to use the concentration of HRP in the total particulate fraction as an estimate for proximal tubular reabsorp-

tion. A constant fractional uptake by the kidney was also reported for other proteins: for serum albumin, ribonuclease, and insulin by Cortney, Sawin, and Weiss [55], for lysozyme by Maack [56,

were reversed, and especially the reabsorption of HRP by the proximal tubule cells was decreased several-fold, when the animals were treated with antagonists to histamine and serotonin, with mannitol, or with hypertonic saline. Relatively low levels of HRP reabsorption also occurred in Wistar-Furth rats in which HRP did not induce vascular leakage as it did in Sprague-Dawley rats [62, 66].

I, in collaboration with Maunsbach, Ottosen, and Pedersen, investigated whether HRP would

57], and for growth hormone by Johnson and Maack [58]. After most of the peroxidase had been cleared

be a good tracer protein for renal micropuncture ex-

from the blood, the concentration of HRP in the total particulate fraction decreased with a half-life of about 7 hours [20]. This decrease probably reflected the slow digestion of HRP by lysosomal cathepsin. A similar rate of breakdown was also reported by Steinman and Cohn [59] for HRP taken up by macrophages in culture. The constancy of fractional uptake of HRP by the renal cortex called to mind the glomerular-tubular balance of sodium reabsorption by the proximal tu-

levels of HRP, the concentration of HRP was measureable accurately in micropuncture samples of 5to 50-nl volumes. It was only necessary to decrease the final volume of the reagent mixture from 3.0 ml (in the usual cuvettes) to 0.11 ml (in a microcuvette of approximately 0.4-ml capacity). The concentration of HRP was measured with O-dianisidine and hydrogen peroxide [59] with a Zeiss, model M4QII, spectrophotometer.

bule cells [60]. It was tested, therefore, whether agents known to affect the reabsorption of sodium would also affect the reabsorption of HRP. Subcutaneous injection of mannitol or intravenous injection of hypertonic saline caused an approximately

ten-fold decrease in the cortical reabsorption of HRP (concentration in the total particulate fractions) [611. The biochemical analysis was corroborated by cytochemical observations. The drastic effects of mannitol or hypertonic saline were difficult to understand until it was observed that antagonists to histamine and serotonin also decreased the uptake of HRP by the renal cortex considerably [621.

It was known from studies by Cotran and Karnovsky [63] that HRP caused vascular leakage in rats due to the release of histamine and serotonin from mast cells. These changes were shown by Deiman, Taugner, and Fahimi [641 to be accompanied by a temporary decrease in the blood pressure. Other changes observed after the injection of HRP were a decrease in the urinary excretion of sodium and an accumulation of HRP in the peritubular spaces surrounding the proximal tubule cells [62, 651. The vascular leakage could be recognized readily by the in-

crease in the hematocrit and the decrease in the plasma protein concentration. Thus, a greatly increased reabsorption of HRP by the proximal tubule cells was accompanied by the following other changes: vascular leakage, a decrease in blood pressure, a decrease in the urinary excretion of sodium, and a rise in the hematocrit. Most of these changes

periments. It was observed that at the usual dose

Thus, HRP offers the possibility to study the reabsorption and excretion of a protein in micropuncture samples by simple spectrophotometric methods. It allows the correlation of micropuncture experiments with the localization of the protein in

the same kidney by cytochemical methods. By

combining the micropuncture analysis of HRP with that of inulin and with the quantitative recovery of the nonreabsorbed HRP from the ureter, it should be possible to find the answers to some, still unresolved questions. Reprint requests to Dr. W. Straus, Department qfBiochemist,y, University of Health Sciences, The Chicago Medical School. 2020 West Ogden Avenue, Chicago, Illinois 60612, USA

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