J. Tiss. Cuh. Me~.13:191-194,1991 1991Tissue Culture Association 0271-8057/91 $01.

50+0,00

QUANTITATION OF FLUID-PHASE ENDOCYTOSIS BY PROXIMAL TUBULAR CELLS IN CULTURE

Mohan I. Abraham and Stephen A. Kempson I

Department of Physiology and Biophysics, Indiana UniversitySchool of Medicine, Indianapolis, Indiana 46202

SUMMARY: Two procedures are described for quantitating fluid-phase endocytosis in monolayer cultures of opossum kidney epithelial cells. Both procedures use the same principle which is to determine uptake of a specific, easily detectable compound in the extracellular fluid. One method uses lucifer yellow, the other uses horseradish peroxidase.

Key words:lucifer yellow; horseradish peroxidase; kidney; fluid-phase endocytosis.

I.

INTRODUCTION
There is good evidence that endocytosis is an important activity of proximal tubule cells in the mammalian kidney. Studies on renal handling of proteins have shown that the proximal tubule is the major site for reabsorption of filtered proteins and that the initial step is endocytosis at the plasma membrane facing the tubule lumen (2,12). Certain peptide hormones, such as insulin, also are internalized by endocytosis without significant extracellular hydrolysis (5). The opossum kidney (OK) cell line (11) is an established line of cells which shares many characteristics of the renal proximal tubule (13,14,16). OK cells have been used to study mechanisms of hormonal regulation of transport processes (1,6,17,22) and intracellular processing of biologically active peptides, including insulin (18) and parathyroid hormone (21). Our recent studies indicate that monolayer cultures of OK cells display endocytic activity (6,10), suggesting that OK cells may be a useful model for detailed studies of endocytosis, and perhaps membrane recycling, in proximal tubule cells. A reliable and straightforward procedure for quantitating fluid-phase endocytosis in OK cells is described. This procedure has been applied successfully to other renal epithelial cell types (9,10) and may be generally applicable to any cell type grown in monolayer culture (4). The principle is to allow the cells to internahze a soluble compound which acts as a "marker" for the extracellular fluid. The amount of fluid-phase marker that is internalized is directly proportional to the rate of fluid-phase endocytosis. Two different fluid-phase markers are compared. These are lucifer yellow (LY), detected by fluores-

cence, and horseradish peroxidase (HRP), detected by a sensitive enzyme assay.

II.

MATERIALS

1To whom correspondenceshould be addressed at Medical Sciences 374, 635 BarnhillDrive, Indianapolis,IN 46202. 191

A. Equipment Fluorescence spectrophotometer, LS-5, Perkin-Elmer1 Spectrophotometer, DU-40, Beckman z High-speed, benchtop microcentrifuge, Eppendorf model 5415, Brinkman 3 Digital dispenser (Volac, 0.5 to 5.0 ml), with 400 ml reservoir, no. P-0875-B, Den~lte 4 Rocker platform, IKA-VIBRAX-VXR model, Janke & Kunkel5 B. Chemicals, solutions, and supphes 1. Lucifer yellow method Lucifer yellow solution, 1.0 mg/ml, prepared from: LY CH, lithium salt, no. L-453, Molecular Probes6 Serum-free culture medium containing 0.1% bovine serum albumin (BSA), fatty-acid free, no. A-6003, Sigma7 Filter sterilize and store frozen in aliquots at - 2 0 C Isotonic washing solution, pH 7.4, prepared from: 145 mM NaC1, no. S-671, Fishers 10 mM Trizma base, no. T-15037 Double distilled water Adjust pH by addition of HEPES, no. H-33757 Perchloric acid, 10.0% (vol/vol), prepared from: Perchloric acid, 60%, no. A-2288 Double distilled water 2. Horseradish peroxidase method Horseradish peroxidase solution, 10.0 mg/ml, prepared from: HRP, type II, no. P-82507 Serum-free culture medium containing 0.1% BSA Filter sterilize and store frozen in aliquots at -20 C

192

ABRAHAM AND KEMPSON Potassium phosphate buffer, pH 5.0, prepared from: Potassium phosphate monobasic, no. P-284 s Double distilled water Adjust pH by addition of sodium hydroxide, no. S-318 s o-Dianisidine solution, 1.0% (wt/vol), prepared from: o-Dianisidine, no. D-91437 (possible carcinogen) Absolute methanol, no. A-452-4 s Make a fresh solution daily and protect from light Hydrogen peroxide solution, 0.3% (vol/vol), prepared from: HRP, 30%, no. H-10097 Double distilled water HRP assay reagent, prepared daily from: o-Dianisidine solution, 0.5 ml Hydrogen peroxide solution, 0.6 ml Potassium phosphate buffer, 60.0 ml Triton solution, 0.05%, prepared from: Triton, no. X-1007 Double distilled water Isotonic washing solution, as above Disposable clear polystyrene cuvettes, 1 cm path length, 1.6 ml vol, no. 67.742, Sarstedt 9 . Measure fluorescence of the acid extracts as soon as possible, using wavelengths of 430 nm for excitation and 540 nm for emission. Subtract tile blank values (usually very low) from all sample readings. . Prepare a standard curve by measuring the fluorescence of LY solutions with concentrations in the range 20 to 200 ng LY/ml. The standard solutions should contain 10% perchloric acid and are made by appropriate dilutions of the stock LY solution. At this concentration perchloric acid does not alter the fluorescence properties of LY (Kempson and Montrose, unpublished observations). . In separate replicate cultures, determine the protein content using the modified Lowry procedure described previously (13,14). 9. Use the LY standard curve to convert sample fluorescence to LY concentration. Express cell uptake of LY in micrograms per milligram protein per 30 rain. B. Horseradish peroxidase method 1. Incubate the cell monolayers in serum-free medium as described above. Adjust the volume of medium to 900 gl at the start of the experiment. 2. Begin by adding 100 #l of the stock HRP solution to each dish. Place on a rocker platform for 2 rain to ensure complete mixing. The final HRP concentration is 1.0 mg/ml. Return the cells to the CO2 incubator at 37 C for 30 min. 3. At the end of the incubation period, remove the medium by aspiration and wash the monolayer 8 times with 2.0-ml aliquots of ice-cold washing solution. Washing steps should be carried out over a period of 10 min. Brief incubation of the monolayer in each aliquot of washing solution will increase removal of HRP which is bound to the surfaces of the cells and the plastic dish. 4. Add 1.0 ml of 0.05% Triton solution to each dish to solubihze the washed monolayer and to release intracellular HRP. Agitate gently on a rocker platform for 30 rain. Separate samples of the cell lysate are used for determination of protein content by the modified Lowry procedure (13,14) and for measurement of HRP activity (see below). 5. Include blanks to correct for surface bound HRP which is not removed by the washing steps. A small amount of HRP always remains attached to the plastic culture surface. The procedure for blanks is to terminate incubation of cells with HRP immediately (within 5 s) after addition of the HRP solution. At this very short time interval there is negligible endocytic uptake of HRP. 6. Horseradish peroxidase activity is determined by placing 50 #1 of cell lysate in a disposable cuvette, followed by 950 #I of HRP assay reagent. Add the reagent rapidly to ensure efficient mixing, record the time, and place the cuvette immediately in the spectrophotometer. After 1.0 min has elapsed, record the absorbance at 460 nm. The absorbance should increase linearly with time for up to 3.0 rain. Check this to ensure that the 1.O-rain time point lies within the linear range.

III.

PROCEDURE

The following procedures are designed for use with OK cell monolayers grown as substrate-dependent monolayers in plastic 35-ram culture dishes. A. Lucifer yellow method: 1. At least 2 h before the experiment, replace the normal culture medium with serum-free medium containing 0.1% BSA. Adjust the medium volume to 950 #1 at the start of the experiment. 2. Begin the assay by adding 50 gl of stock LY solution to each dish, and place on a rocker platform for 2 rain to ensure complete mixing. The final LY concentration is 0.05 mg/ml. Return the cells to the CO2 incubator at 37 C for 30 rain. 3. After completion of the incubation with LY, remove the medium by aspiration, and rapidly but carefully wash the monolayer 6 times with 2.0-ml aliquots of ice-cold washing solution. The digital dispenser is highly recommended. Intracellular LY is extracted by addition of 1.0 ml of 10% perchloric acid. Leave at room temperature for 15 rain without agitation. 4. Blanks should be included to correct for trapping and surface binding of LY to the cells. In these dishes, the incubation of cells with LY is terminated immediately (within 5 s) after addition of LY solution. There is negligible endocytic uptake of LY at this very short time interval. 5. Remove the perchloric acid extract from each dish and transfer to a 1.5-ml microcentrifuge tube. Centrifuge at 13 000 Xg for 5 rain to sediment any cell debris that is present. Centrifugation is usually unnecessary because the acid-insoluble material tends to stick to the plastic culture surface.

ABRAHAM AND KEMPSON 7. Subtract the blank values from all sample readings. Values for the blanks should be less than 10% of the sample values. 8. Determine HRP activity in standard solutions with concentrations in the range 0.1 to 1.0 gg HRP/ml. The standards are made by appropriate dilutions with water of the HRP stock solution. 9. Use the HRP standard curve to convert absorbance to concentration of HRP. Express cell uptake of HRP in microgram per milligram protein per 30 min.

193

c-

E
O 09
t,.,,,t

IV.

DISCUSSION
Lucifer yellow and HRP are used as fluid-phase markers because they are readily soluble, not cytotoxic, easily detectable, there is negligible binding to the plasma membrane, and extraceUular degradation is minimal. Evidence that these markers are taken up primarily by fluidphase endocytosis into OK ceils is provided by several properties of the uptake process. One of the most important observations is that the uptake process is not saturable at concentrations in the milligram per milliliter range (19). This is true for both LY and HRP in OK cells. Uptake of LY at 37" C is not saturable and increases steadily over the concentration range 0.03 to 0.70 mg/ml (Fig. 1). HRP uptake shows a similar, almost linear increase over the range 0.1 to 1.0 mg/ml (6). The nonsaturable nature of the uptake process indicates that receptor-mediated endocytosis, or a specific plasma membrane transport system, is not invok, ed. Another important observation is the dependence of the uptake process on a metabolic energy supply. Thus, pre0.6

(1) O

E
o..
rr" "1:a.

O
FiG. 2. Uptake of HRP by OK cell monolaycrs is decreased in cells precooled and maintained at 4 C. Data are mean values from a typical experiment analyzed in triplicate.

-_=
,o

0.4

E
:a.

m 'v'
:2)

0.2

0.2

0.4

0.6

0.8

LY CONCENTRATION, mg/ml
FIG. 1. Uptake of LY by OK cell monolayers at different extracellular concentrations of LY. At 37 C cell uptake of LY is not saturable. Uptake of LY is markedly decreased in cells precooled and maintained at 4 C. Data are mean values from a typical experiment analyzed in triplicate.

cooling OK cells to 4 C produces a marked reduction in cell uptake of both LY (Fig. 1) and HRP (Fig. 2). Note that for studies on temperature dependence the ceils are incubated in an isotonic inorganic salt solution buffered with HEPES, pH 7.4 (6) to avoid the need for a CO 2 incubator. Similarly, metabolic inhibitors such as KCN also produce inhibition of uptake of both LY and HRP (8). Demonstrated dependence on an energy supply rules out the possibility that the fluid-phase markers enter the cells by nonendocytic pathways such as diffusion. A maior advantage of using LY is the relatively simple procedure for quantitating intraceilular LY. Furthermore, the fluorescence properties of the dye allow a rapid visual check of the cells under a fluorescence microscope. The intracellular distribution of LY is typically non-uniform and punctate, as would be expected if the dye is contained within endocytic vesicles (7). A potential problem can arise if LY is used to measure endocytosis after drug action or chemical modifications designed primarily to interfere with the endocytic process. The metabolic inhibitor iodoacetate, for example, produces a marked increase in OK cell uptake of LY. We attribute this increase to

194

ABRAHAM AND KEMPSON

enhanced LY uptake by nonendocytic pathways (8). This is probably due to secondary effects of iodoacetate which alter the permeability properties of the plasma membrane toward small molecules such as LY [molecular weight (Mr) 457]. Horseradish peroxidase is a much larger molecule (M r 40 000) and cell uptake of HRP is unlikely to be influenced by the kind of permeability changes discussed above. Indeed, iodoacetate produces marked inhibition of HRP uptake by OK cells, as expected (8). Another advantage of HRP is the availability of a histochemical procedure to produce a reaction product which can be observed by electron microscopy. This allows precise determination that intracellular HRP is confined to the interior of apical endocytic vesicles (6). Although there is a procedure for visualizing LY in the electron microscope (15), the method has not been used widely for studies on endocytosis. Growth of renal epithelial cells on permeable supports allows the study of transcytosis, i.e. the vectorial transfer of endocytosed material from the apical to the basal side of the cell, or vice-versa. Both LY and HRP have been used for quantitating transcytosis in primary cultures of proximal tubule cells (3) and in Madin-Darby canine kidney (MDCK) cells (20). V. REFERENCES

7.

8. 9.

10.

1 t.

12.

13.

14.

15. 16.

17.

1. Abraham, M. I.; McAteer, J. A.; Kempson, S. A. Insulin stimulates phosphate transport in opossum kidney epithelial cells. Am. J. Physiol. 258:F1592-F1598; 1990. 2. Coudrier, E.; Kerjaschki, D.; Louvard, D. Cytoskeletal organization and submembranous interactions in intestinal and renal brush borders. Kidney Int. 34:309-320; 1988. 3. Goligorsky, M. S.; Hruska, K. A. Transcytosis in cultured proximal tubular cells. J. Membr. Biol. 93:237-247; 1986. 4. Guillot, F. L.; Audus, K. L.; Raub, T. J. Fluid-phase endocytosis by primary cultures of bovine brain microvessel endothelial cell monolayers. Microvasc. Res. 39:1-14; 1990. 5. Hjelle, J. T.; Oparil, S.; Peterson, D. R. Subceltular sites of insulin hydrolysis in renal proximal tubules. Am. J. Physiol. 246:F409-F416; 1984. 6. Kempson, S. A.; Helmle, C.; Abraham, M. I., et al. Parathyroid hormone

18.

19. 20.

21.

22.

action on phosphate transport is inhibited by high osmolality. Am. J. Physiol. 258:F1336-F1344; 1990. Kempson, S. A.; Helmle, C.; Murer, H. Endocytosis and phosphate transport in OK epithelial cells. Renal Physiol. Biochem. 12:359-364; 1989. Kempson, S. A.; Kunkler, K. J.; Murer, H. Iodoacetate action on iluidphase endocytosis in OK cells. Physiologist 32:212; 1989 (Abstract). Kempson, S. A.; McAteer, J. A.; AI-Mahrouq, H., et al. Proximal tubule characteristics of cultured human renal cortex epithelium. J. Lab. Clln. Med. 113:285-296; 1989. Kempson, S. A.; Ying, A. L.; McAteer, J. A., et al. Endocytosis and Na+/solute cotransport in renal epithelial ceils. J. Biol. Chem. 264:18451-18456; 1989. Koyama, H.; Goedpasture, C.; Miller, M. M., et al. Establishment and characterization of a celt line from the American opossum (Didelphys virginiana). In Vitro 14:239-246; 1978. Maack, T.; Park, C. H.; Camargo, M. J. F. Renal filtration, transport, and metabolism of proteins. In: Seldin, D. W.; Giebisch, G., eds. The kidney: physiology and pathophysiology. New York: Raven Press; 1985:1773-1803, Malmstrom, K.; Murer, H. Parathyroid hormone inhibits phosphate transport in OK cells but not in LLC-PK1 and JTC12.P3 cells. Am. J. Physiol. 251:C23-C31; 1986. Malmstrom, K.; Stange, G.; Murer, H. Identification of proximal tubular transport functions in the established kidney cell line, OK. Biochim. Biophys. Acta 902:269-277; 1987. Maranto, A. R. Neuronal mapping: a photooxidation reaction makes lucifer yellow useful for electron microscopy. Science 217:953-955; 1982. Pollock, A. S.; Warnock, D. G.; Strewler, G. J. Parathyroid hormone inhibition of Na+-H+ antiporter activity in a cultured renal cell line. Am. J. Physiol. 250:F217-F225; 1986. Quamme, G.; Biber, J.; Muter, H. Sodium-phosphate cotransport in OK cells: inhibition by PTH and "'adaptation" to low phosphate. Am. J. Physiol. 257:F967-F973, 1989. Rabkiu, R.; Yagil, C.; Frank, B. Basolateral and apical binding, internalization, and degradation of insulin by cultured kidney epithelial cells. Am. J. Physiol. 257:E895-E902; 1989. Steinman, R. M.; Mellman, I. S.; Muller, W. A., et al. Endocytosis and the recycling of plasma membrane. J. Cell Biol. 96:1-27; 1983. Van Bonsdorf, C.-H.; Fuller, S. D.; Simons, K. Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters. EMBO J. 4:2781-2792; t985. Yamaguchi, T.; Fukase, M.; Nishikawa, M., et al. Parathyroid hormone degradation by chymotrypsin-like endopeptidase in the opossum kidney cell. Endocrinology 123:2812-2817; 1988. Yonemura, K.; Cheng, L.; Sacktor, B., et al. Stimulation by thyroid hormone of Na+-H+ exchange activity in cultured opossum kidney cells. Am. J. Physiol. 258:F333-F338; 1990.

We are indebted to Dr. M. H. Montrose (Johns Hopkins University, Baltimore) and Prof. H. Murer (University of Zurich, Switzerland) for helpful discussions and suggestions during development of the methodology described here. M. I. A. was supported by a Postdoctoral Fellowship from the American Heart Association, Indiana Aifihate Inc. S. A. K. was supported by a Research Career Development Award and grant DK32148 from the National Institutes of Health.

1 Perkin-Elmer Corp., Norwalk, CT 2 Beckman Instruments Inc., Spinco Division, Palo Alto, CA 3 Brinkmau Instruments Inc., Westbury, NY 4 Denville Scientific Inc., Denville, NJ 5 Janke & Kunkel GmbH., Staufen, W. Germany

6 Molecular Probes Inc., Eugene, OR 7 Sigma Chemical Co., St. Louis, MO s Fisher Scientific, Pittsburgh, PA 9 Sarstedt Inc., Princeton, NJ