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[66] Isolation of renal tubules
658
ISOLATION AND CULTURE OF CELLS
[66]
sible to have more than one size of hole in sieves of the same mesh. Thus both of these 2 sieve characteristics should be observed in this isolation method, and should be considered if it is desired to modify the method for glomerular isolation from kidneys of other species. The method described here has been used for metabolic studies on normal and nephrotoxic nephritic rat glomeruli; modifications of this technique to suit different individual studies have also been reported. 5,9,1°,13 Such studies have delineated metabolic differences between glomeruli and other nephron units, and have provided new understanding of the physiological activities of normal glomeruli and of the pathogenesis of glomerular injury. aSD. G. Taylor, R. G. Price, and D. Robinson, Biochem. ]. 121, 27 (1971).
[66] I s o l a t i o n of R e n a l T u b u l e s B y GEORGES RORIVE and ARNOST KLEINZELLER
For many years, slices of kidney cortex have been used to study the mechanisms of the renal transport system. They have provided numerous and provocative results, but they have definite limitations, principally when kinetic studies are involved. 1,2 These limitations are mainly due to the lack of homogeneity of the tissue (although in cortical slices of rabbit, the major portion of the cells stems from proximal tubules), and the complex geometry of the preparation. Therefore attempts to obtain individual living cells from kidney tissue are important. 1. Bosackova, 3 by gentle trypsinization of kidney cortex slices, obtained a suspension of isolated cells which accumulated p-aminohippuric acid (PAH) and K. In our hands this technique provided only a low yield of cells markedly contaminated with subcellular components. The method could not be improved by treating the tissue with a mixture of enzymes, such as trypsin, hyaluronidase, and collagenase. 2. Thimmappayya et al. 4 described briefly a method for preparing a suspension of renal cells by breaking up mechanically tissue previously 'M. B. Burg and J. Orloff, Amer. J. Physiol. 203, 327 (1962). ~K. C. Huang and D. S. T. Lin, Amer. J. Physiol. 208, 391 (1965). s j. Bosackova, Physiol. Biochem. 11, 39 (1962). 4B. Thimmappayya, R. Ramachandra Reddy, and P. M. Bhargava, Exp. Cell Res. 63, 333 (1970).
[66]
ISOLATION OF RENAL TUBULES
659
impoverished of Ca. Little is known of the properties of this preparation beyond its capability of linearly taking up O., at a rate somewhat lower than that found in isolated tubules. 3. Borle 5 suggested the use of cells grown in vitro (established cell line LL CMK2 derived from rhesus monkey kidneys; available from Microbiological Associates, Bethesda, Maryland). But even if these cells were originally derived from kidney, because of dedifferentiation they do not possess all the metabolic properties of cells derived from cortical nephrons. 6 On account of the absence of satisfactory suspension of isolated kidney cells, the preparation of isolated kidney tubules prepared by the procedure of Burg and Orloff 1 for rabbit kidney cortex and by Nagata and Rasmussen" for rat kidney provide an experimental model with some advantages as compared to slices'~: (1) The diffusion pathway of metabolic substrates to the cells is reduced, thus eliminating problems such as the limiting thickness of the tissue. (2) The peritubular face of the cells is directly accessible to transported solutes. The extent to which the luminal face of renal cells is accessible to solutes remains a matter of conjecture for both slices and isolated tubules. (3) The experimental manipulations with a suspension of tissue material are in many respects easier than with slices. (4) In isolated tubules active transport of K, 1 p-aminohippurate, 2 and sugars ~' was demonstrated. The rate of these processes is about 3-fold higher than in slices. Materials
Collagenase type I (Sigma Co., St. Louis, Missouri) Hyaluronidase type I (Sigma Co., St. Louis, Missouri) Balanced physiological solution (per liter): NaC1, 8.0 g; KC1, 0.4 g; MgSO~.7H~O, 0.2 g; CaC12, 0.14 g; (1) dissolved separately: Na.,HPO4.2H.,O, 0.06 g; KH,,PO4, 0.06 g; NaHCO.~, 0.35 g; glucose,1 g; final pH 7.4 (Hanks and Wallace TM) Calf serum (Flow Laboratories, Rockville, Maryland) Dow Corning antifoam A Surgical gauze (Bauer and Black No. 245).
5A. B. Borle, Endocrinology 85, 194 (1969). ~H. Rasmussen, Amer. J. Med. 50, 567 (1971). 7 N. Nagata and H. Rasmussen, Biochim. Biophys. Acta 215, 1 (1970). 8 E. M. Clarkson, L. B. Talner, and H. E. de Wardener, Clinical Sci. 38, 617 (1970). ~A. Kleinzeller, J. Kolinska, and I. Benes, Biochern. d. 104, 843 (1967). '°J. H. Hanks and R. E. Wallace, Proc. Soc. Exp. Biol. Med. 71, 196 (1949).
660
ISOLATION AND CULTURE OF CELLS
[66]
Isolation Procedure The procedures to obtain isolated tubules from the rabbit or from the rat kidney cortex are somewhat different and will be described successively. Preparation of Isolated Tubules Fragments from Rabbit Kidney Cortex. The procedure used is a modification of the original description by Burg and Ofloff.1 New Zealand White rabbits weighing about 2 kg are killed by a blow on the neck. Kidneys are rapidly exposed, and a needle (No. 2) is tied in place in each renal artery; 10 ml of standard saline physiological solution at 4 ° are slowly injected into both kidneys, first to cool the tissue, and second to wash the blood out. The renal veins are then occluded with the arterial ligatures, and 10-15 ml iced collagenase solution (collagenase 0.3% dissolved in the standard saline solution) are forcibly injected into each kidney until the renal capsule ruptures and fluid escapes through the surface of the kidneys. Kidneys are then removed and kept on ice. The cortex is dissected and diced with small anatomical scissors to obtain fragments approximately 2-3 mm in size. These fragments are placed in a l-liter conical siliconized flask containing 40 ml of collagenase solution, and oxygenated with a O~-CO2 mixture (95:5 v/v). They are incubated for 45-60 minutes, at 25-30 ° with slow mixing by magnetic stirrer. After incubation, the enzymatic action is stopped by addition of 120 ml of cold saline containing 3% calf serum, and the suspension is filtered through three layers of surgical gauze into 50-ml plastic tubes kept on ice. The filtrate is then centrifuged for 90 seconds at 50 g, in a refrigerated Sorvall RC2B centrifuge. The supernatant is removed by suction and discarded. The pellet is resuspended in saline containing 3% calf serum, and centrifuged again. This procedure is repeated three times. The final pellet is resuspended in the experimental saline containing 3% calf serum, in order to obtain a tissue concentration of approximately 25 mg wet weight per milliliter and stored on ice. The yield is usually 500-750 mg of tubules per kidney. The suspension consists principally of short fragments of proximal tubules, some isolated cells and separated glomeruli. Electron microscope examination of cells before and after incubation reveals excellent preservation of cell membranes and organelles. 11 Preparation of Isolated Tubule Fragments from Rat Kidney Cortex. Isolated tubules from rat kidney cortex cannot be obtained by the classical ~IA. Struyvenberg, R. B. Morrison and A. S. Relman, Amer. 1. Physiol. 214, 1155 (1968).
[66]
ISOLATION OF RENAL TUBULES
661
procedure described for rabbit kidney. A valuable preparation can be obtained, however, by the use of a modification of the method proposed by Howard and Pesch TM and by Howard e t al. 13 for the preparation of intact liver cells; the modification was suggested by Nagata and RasmussenY Rats weighing 150-200 g are killed by a blow on the neck. The abdominal aorta is exposed through a midline abdominal incision. The celiac artery, the abdominal aorta, and the inferior vena cava are ligated just below the diaphragm. The kidneys are then perfused via the distal part of the abdominal aorta first with 10 ml of Ca-free physiological solution at 4 ° followed by 10 ml of an enzyme solution containing 40 mg of collagenase per 100 ml, 100 mg of hyaluronidase per 100 ml dissolved in Ca-free physiological solution. The kidneys are rapidly removed and the cortex was dissected free. Cortices from 4 to 6 kidneys are combined, diced with anatomical scissors, and suspended in 10 ml of the enzyme solution in a 250-ml Erlenmeyer flask. This flask is incubated at 37 ° under O~-CO_~ (95:5 v / v ) for 60 minutes with gentle stirring. At the end of the incubation time 30 ml of cold Ca-free physiological solution is added and gently mixed with the enzyme solution. The content of the flask is then filtered through three layers of surgical gauze, into 50-ml centrifuge tubes. Isolated tubules are obtained by centrifugation as described for rabbit kidney cortex. Light and electron microscopic examination reveal that the obtained preparation contains both proximal and distal tubules with basement membranes largely intact and a normal arrangement and configuration of mitochondriaY Incubation Procedure The suspension of tubules (final concentration usually 25 mg wet weight per milliliter) is incubated at 25 ° in special vessels (Fig. 1) and oxygenated with 02 or a mixture of 02 and CO._,, according to the buffer system selected. The incubation flasks are modified from Burg and Orloff ~ as suggested by Struyvenberg et al. 'a and Clarkson e t al. s (Fig. 1). A sintered-glass disk 1 cm in diameter is inserted near the bottom of the tube, ensuring a fine dispersion of gas bubbles. The use of this special incubation vessel practically eliminates cell destruction which is apparent when a conventional shaking apparatus is used. Cell breakage could also occur during transfer of the suspension but is minimized by using serological pipettes with wide tips, or syringes with laboratory cannulas. To avoid 12R. B. Howard and L. A. Pesch, 1. Biol. C h e m . 243, 3105 (1968). 1~R. B. Howard, A. K. Christensen, F. A. Gibbs, and L. A. Pesch, 35, 675 (1967).
J. Cell Biol.
662
ISOLATION A N D C U L T U R E OF CELLS
i• i
[66]
02
Tubul~ Suspension
I
Constant-temperature balh
Fro. 1. Special incubation flask used for the tubule suspension. foaming during incubation, the inside of the flask was sprayed with Dow Corning antifoam A prior to use. Sampling Procedure Standard Procedure
A sample, usually 2 ml, of the well-homogenized tubule suspension is removed from the incubation flask, using serological pipette or syringe, and poured into special centrifugation tubes, as described by Burg and Orloff 1 (Fig. 2). These tubes are centrifuged at 15,000 g, for 10 minutes in a refrigerated Sorvall RC2B centrifuge, using an HB4 Sorvall horizontal rotor. The supernatant and the white top layer of cells are removed by suction. For Water and Ionic Content Determination. The pellet of tubules is directly ejected with a stainless steel stylet from the centrifugation tube into a Teflon vial and immediately weighed. Tissue water content is determined by drying the tissue to constant weight in a vacuum oven at 90 °. For Na, K, C1, Ca, and Mg content, the dry tissue is extracted for 2 days, at room temperature in 5 ml of 0.1 N HNO3, and the supernatant is diluted for determination of ion content by atomic absorption photometry. Tritiated inulin (New England Nuclear) is used to estimate the incubation medium trapped inside the plug of tubules. ~H is determined in both the medium and the 0.1 N HNO3 tissue extract. One milliliter of an adequate dilution is counted in 10 ml of scintillation solution: toluene, 2 liters; Triton X 100, 1 liter; Omnifluor (New England Nuclear), 12 g.14 ~4G. Rorive, R. Mielsen, and A. K1einzeller, Biochim. Biophys. Acta 266, 376 (1972).
[66]
ISOLATION OF RENAL TUBULES
4
663
12mrr
i
i Lucite E E o
Medium
E E
@ _
.
.
.
.
.
iiiiij t
.
Pocked lubules
"Teflon plug 2ram
FIG. 2. Special centrifuge tube used to separate tubules from incubation medium.
For Sugar-Uptake Studies. The plug of tubules, after wet weight determinations is placed in homogenizing tubes containing 3 ml of 1.67% ZnSO,.7H20 (w/v). The tissue was then homogenized, and 1 ml of previously titrated 0.15 M Ba(OH)2 is added. The tubes are centrifuged, and the supernatants are used for sugar determination. For p-Aminohippurate (PAH)-Uptake Studies. The pellet of tubules is directly suspended in 2 ml of 3% trichloroacetic acid (w/v). Protein precipitate is removed by centrifugation, and PAH concentration is measured on the supernatant. ~s For Amino Acid-Uptake Studies. Hillman et al. a6 proposed taking a 2-ml sample of the tubule suspension, cooling it rapidly to 0 °, and centrifuging it at 48,000 g for 8 minutes. The medium is removed, and the cell pellet is washed twice with ice-cold physiological saline solution. The sides of the tube and the surface of the pellet are dried by suction. The 15M. B. Burg and J. Orloff, Amer. J. Physiol. 217, 1064 (1969). 16R. E. Hillman, I. Albrecht, and L. E. Rosenberg, ]. Biol. Chem. 243, 5566 (1968).
66 4
ISOLATION AND CULTURE OF CELLS
[66]
renal tubules are lysed by adding 2 ml of distilled water and boiling for 10 minutes. Amino acids are determined on the clear supernatant. Kinetic Studies
The above procedure can be used for determination of the steady-state ionic and water content, or of the steady-state accumulation of sugar, amino acid, or p-aminohippurate, but has to be modified for kinetic studies. For 24Na and 42K flux studies, Burg e t al. 17 have proposed a faster sampling procedure. Rapid separation of tubules from the medium can be achieved by placing 1-4 ml of the medium suspension in a BiJchner funnel (inside diameter 55 mm) fitted with filter paper (Whatman No. 541) and filled with 50 ml of ice-cold Na- and K-free diluent, for instance, trihydroxymethylaminomethane (Tris) 155M or sucrose, 310M. Suction is immediately applied, and the filter paper is washed twice with 25 ml of ice-cold Na-, K-flee diluent. Although the procedure requires approximately 15 seconds, Burg e t al. 17 suggest that, at least for ~4Na and 42K, exchange of isotope between the medium and the tissue appears to be terminated immediately on addition of the suspension to the diluent. Filter papers containing the tissue are extracted overnight with 10 ml of 0.75 N.HNO3. Radioactivity is assayed in 1 ml of this extract, and ionic content is measured on the same extract by atomic absorption photometry. To estimate the amount of radioactivity and ions trapped in the filter paper, an amount of radioactive medium equal in volume to the amount of suspension is introduced into the Biichner funnel. The filter paper is washed, extracted, and analyzed for radioactivity and ionic content in the same manner as for the suspension. The amount of ions and radioactivity found trapped in the filter paper is subtracted from the experimental values. These corrections are usually lower than 5 % for Na and less than 1% for K, and thus do not have an important effect on the final results. The amount of cations recovered from tissues after filtration is approximately 60% of the original content of the tissue in an identical volume of suspension. Burg e t al., 1~ however, have well established the fact that the fraction of ions recovered from the filter paper is representative of that present in the tissue. The ratio Na:K is the same in extract of tissue removed by filtration or by centrifugation.1~ Presumably, the loss of cations during filtration could be related to the passage of cells through or around the filter or to cell damage during handling. Murthy and Foulkes TM have used a quite similar procedure to study the movements of PAH labeled with '4C. i'M. B. Burg, E. F. Grollman, and J. Orloff, Amer. J. Physiol. 206, 483 (1964). "SL. Murthy and E. C. Foulkes, Nature (London) 213, 180 (1967).
ISOLATION AND CULTURE OF CELLS
[66]
sible to have more than one size of hole in sieves of the same mesh. Thus both of these 2 sieve characteristics should be observed in this isolation method, and should be considered if it is desired to modify the method for glomerular isolation from kidneys of other species. The method described here has been used for metabolic studies on normal and nephrotoxic nephritic rat glomeruli; modifications of this technique to suit different individual studies have also been reported. 5,9,1°,13 Such studies have delineated metabolic differences between glomeruli and other nephron units, and have provided new understanding of the physiological activities of normal glomeruli and of the pathogenesis of glomerular injury. aSD. G. Taylor, R. G. Price, and D. Robinson, Biochem. ]. 121, 27 (1971).
[66] I s o l a t i o n of R e n a l T u b u l e s B y GEORGES RORIVE and ARNOST KLEINZELLER
For many years, slices of kidney cortex have been used to study the mechanisms of the renal transport system. They have provided numerous and provocative results, but they have definite limitations, principally when kinetic studies are involved. 1,2 These limitations are mainly due to the lack of homogeneity of the tissue (although in cortical slices of rabbit, the major portion of the cells stems from proximal tubules), and the complex geometry of the preparation. Therefore attempts to obtain individual living cells from kidney tissue are important. 1. Bosackova, 3 by gentle trypsinization of kidney cortex slices, obtained a suspension of isolated cells which accumulated p-aminohippuric acid (PAH) and K. In our hands this technique provided only a low yield of cells markedly contaminated with subcellular components. The method could not be improved by treating the tissue with a mixture of enzymes, such as trypsin, hyaluronidase, and collagenase. 2. Thimmappayya et al. 4 described briefly a method for preparing a suspension of renal cells by breaking up mechanically tissue previously 'M. B. Burg and J. Orloff, Amer. J. Physiol. 203, 327 (1962). ~K. C. Huang and D. S. T. Lin, Amer. J. Physiol. 208, 391 (1965). s j. Bosackova, Physiol. Biochem. 11, 39 (1962). 4B. Thimmappayya, R. Ramachandra Reddy, and P. M. Bhargava, Exp. Cell Res. 63, 333 (1970).
[66]
ISOLATION OF RENAL TUBULES
659
impoverished of Ca. Little is known of the properties of this preparation beyond its capability of linearly taking up O., at a rate somewhat lower than that found in isolated tubules. 3. Borle 5 suggested the use of cells grown in vitro (established cell line LL CMK2 derived from rhesus monkey kidneys; available from Microbiological Associates, Bethesda, Maryland). But even if these cells were originally derived from kidney, because of dedifferentiation they do not possess all the metabolic properties of cells derived from cortical nephrons. 6 On account of the absence of satisfactory suspension of isolated kidney cells, the preparation of isolated kidney tubules prepared by the procedure of Burg and Orloff 1 for rabbit kidney cortex and by Nagata and Rasmussen" for rat kidney provide an experimental model with some advantages as compared to slices'~: (1) The diffusion pathway of metabolic substrates to the cells is reduced, thus eliminating problems such as the limiting thickness of the tissue. (2) The peritubular face of the cells is directly accessible to transported solutes. The extent to which the luminal face of renal cells is accessible to solutes remains a matter of conjecture for both slices and isolated tubules. (3) The experimental manipulations with a suspension of tissue material are in many respects easier than with slices. (4) In isolated tubules active transport of K, 1 p-aminohippurate, 2 and sugars ~' was demonstrated. The rate of these processes is about 3-fold higher than in slices. Materials
Collagenase type I (Sigma Co., St. Louis, Missouri) Hyaluronidase type I (Sigma Co., St. Louis, Missouri) Balanced physiological solution (per liter): NaC1, 8.0 g; KC1, 0.4 g; MgSO~.7H~O, 0.2 g; CaC12, 0.14 g; (1) dissolved separately: Na.,HPO4.2H.,O, 0.06 g; KH,,PO4, 0.06 g; NaHCO.~, 0.35 g; glucose,1 g; final pH 7.4 (Hanks and Wallace TM) Calf serum (Flow Laboratories, Rockville, Maryland) Dow Corning antifoam A Surgical gauze (Bauer and Black No. 245).
5A. B. Borle, Endocrinology 85, 194 (1969). ~H. Rasmussen, Amer. J. Med. 50, 567 (1971). 7 N. Nagata and H. Rasmussen, Biochim. Biophys. Acta 215, 1 (1970). 8 E. M. Clarkson, L. B. Talner, and H. E. de Wardener, Clinical Sci. 38, 617 (1970). ~A. Kleinzeller, J. Kolinska, and I. Benes, Biochern. d. 104, 843 (1967). '°J. H. Hanks and R. E. Wallace, Proc. Soc. Exp. Biol. Med. 71, 196 (1949).
660
ISOLATION AND CULTURE OF CELLS
[66]
Isolation Procedure The procedures to obtain isolated tubules from the rabbit or from the rat kidney cortex are somewhat different and will be described successively. Preparation of Isolated Tubules Fragments from Rabbit Kidney Cortex. The procedure used is a modification of the original description by Burg and Ofloff.1 New Zealand White rabbits weighing about 2 kg are killed by a blow on the neck. Kidneys are rapidly exposed, and a needle (No. 2) is tied in place in each renal artery; 10 ml of standard saline physiological solution at 4 ° are slowly injected into both kidneys, first to cool the tissue, and second to wash the blood out. The renal veins are then occluded with the arterial ligatures, and 10-15 ml iced collagenase solution (collagenase 0.3% dissolved in the standard saline solution) are forcibly injected into each kidney until the renal capsule ruptures and fluid escapes through the surface of the kidneys. Kidneys are then removed and kept on ice. The cortex is dissected and diced with small anatomical scissors to obtain fragments approximately 2-3 mm in size. These fragments are placed in a l-liter conical siliconized flask containing 40 ml of collagenase solution, and oxygenated with a O~-CO2 mixture (95:5 v/v). They are incubated for 45-60 minutes, at 25-30 ° with slow mixing by magnetic stirrer. After incubation, the enzymatic action is stopped by addition of 120 ml of cold saline containing 3% calf serum, and the suspension is filtered through three layers of surgical gauze into 50-ml plastic tubes kept on ice. The filtrate is then centrifuged for 90 seconds at 50 g, in a refrigerated Sorvall RC2B centrifuge. The supernatant is removed by suction and discarded. The pellet is resuspended in saline containing 3% calf serum, and centrifuged again. This procedure is repeated three times. The final pellet is resuspended in the experimental saline containing 3% calf serum, in order to obtain a tissue concentration of approximately 25 mg wet weight per milliliter and stored on ice. The yield is usually 500-750 mg of tubules per kidney. The suspension consists principally of short fragments of proximal tubules, some isolated cells and separated glomeruli. Electron microscope examination of cells before and after incubation reveals excellent preservation of cell membranes and organelles. 11 Preparation of Isolated Tubule Fragments from Rat Kidney Cortex. Isolated tubules from rat kidney cortex cannot be obtained by the classical ~IA. Struyvenberg, R. B. Morrison and A. S. Relman, Amer. 1. Physiol. 214, 1155 (1968).
[66]
ISOLATION OF RENAL TUBULES
661
procedure described for rabbit kidney. A valuable preparation can be obtained, however, by the use of a modification of the method proposed by Howard and Pesch TM and by Howard e t al. 13 for the preparation of intact liver cells; the modification was suggested by Nagata and RasmussenY Rats weighing 150-200 g are killed by a blow on the neck. The abdominal aorta is exposed through a midline abdominal incision. The celiac artery, the abdominal aorta, and the inferior vena cava are ligated just below the diaphragm. The kidneys are then perfused via the distal part of the abdominal aorta first with 10 ml of Ca-free physiological solution at 4 ° followed by 10 ml of an enzyme solution containing 40 mg of collagenase per 100 ml, 100 mg of hyaluronidase per 100 ml dissolved in Ca-free physiological solution. The kidneys are rapidly removed and the cortex was dissected free. Cortices from 4 to 6 kidneys are combined, diced with anatomical scissors, and suspended in 10 ml of the enzyme solution in a 250-ml Erlenmeyer flask. This flask is incubated at 37 ° under O~-CO_~ (95:5 v / v ) for 60 minutes with gentle stirring. At the end of the incubation time 30 ml of cold Ca-free physiological solution is added and gently mixed with the enzyme solution. The content of the flask is then filtered through three layers of surgical gauze, into 50-ml centrifuge tubes. Isolated tubules are obtained by centrifugation as described for rabbit kidney cortex. Light and electron microscopic examination reveal that the obtained preparation contains both proximal and distal tubules with basement membranes largely intact and a normal arrangement and configuration of mitochondriaY Incubation Procedure The suspension of tubules (final concentration usually 25 mg wet weight per milliliter) is incubated at 25 ° in special vessels (Fig. 1) and oxygenated with 02 or a mixture of 02 and CO._,, according to the buffer system selected. The incubation flasks are modified from Burg and Orloff ~ as suggested by Struyvenberg et al. 'a and Clarkson e t al. s (Fig. 1). A sintered-glass disk 1 cm in diameter is inserted near the bottom of the tube, ensuring a fine dispersion of gas bubbles. The use of this special incubation vessel practically eliminates cell destruction which is apparent when a conventional shaking apparatus is used. Cell breakage could also occur during transfer of the suspension but is minimized by using serological pipettes with wide tips, or syringes with laboratory cannulas. To avoid 12R. B. Howard and L. A. Pesch, 1. Biol. C h e m . 243, 3105 (1968). 1~R. B. Howard, A. K. Christensen, F. A. Gibbs, and L. A. Pesch, 35, 675 (1967).
J. Cell Biol.
662
ISOLATION A N D C U L T U R E OF CELLS
i• i
[66]
02
Tubul~ Suspension
I
Constant-temperature balh
Fro. 1. Special incubation flask used for the tubule suspension. foaming during incubation, the inside of the flask was sprayed with Dow Corning antifoam A prior to use. Sampling Procedure Standard Procedure
A sample, usually 2 ml, of the well-homogenized tubule suspension is removed from the incubation flask, using serological pipette or syringe, and poured into special centrifugation tubes, as described by Burg and Orloff 1 (Fig. 2). These tubes are centrifuged at 15,000 g, for 10 minutes in a refrigerated Sorvall RC2B centrifuge, using an HB4 Sorvall horizontal rotor. The supernatant and the white top layer of cells are removed by suction. For Water and Ionic Content Determination. The pellet of tubules is directly ejected with a stainless steel stylet from the centrifugation tube into a Teflon vial and immediately weighed. Tissue water content is determined by drying the tissue to constant weight in a vacuum oven at 90 °. For Na, K, C1, Ca, and Mg content, the dry tissue is extracted for 2 days, at room temperature in 5 ml of 0.1 N HNO3, and the supernatant is diluted for determination of ion content by atomic absorption photometry. Tritiated inulin (New England Nuclear) is used to estimate the incubation medium trapped inside the plug of tubules. ~H is determined in both the medium and the 0.1 N HNO3 tissue extract. One milliliter of an adequate dilution is counted in 10 ml of scintillation solution: toluene, 2 liters; Triton X 100, 1 liter; Omnifluor (New England Nuclear), 12 g.14 ~4G. Rorive, R. Mielsen, and A. K1einzeller, Biochim. Biophys. Acta 266, 376 (1972).
[66]
ISOLATION OF RENAL TUBULES
4
663
12mrr
i
i Lucite E E o
Medium
E E
@ _
.
.
.
.
.
iiiiij t
.
Pocked lubules
"Teflon plug 2ram
FIG. 2. Special centrifuge tube used to separate tubules from incubation medium.
For Sugar-Uptake Studies. The plug of tubules, after wet weight determinations is placed in homogenizing tubes containing 3 ml of 1.67% ZnSO,.7H20 (w/v). The tissue was then homogenized, and 1 ml of previously titrated 0.15 M Ba(OH)2 is added. The tubes are centrifuged, and the supernatants are used for sugar determination. For p-Aminohippurate (PAH)-Uptake Studies. The pellet of tubules is directly suspended in 2 ml of 3% trichloroacetic acid (w/v). Protein precipitate is removed by centrifugation, and PAH concentration is measured on the supernatant. ~s For Amino Acid-Uptake Studies. Hillman et al. a6 proposed taking a 2-ml sample of the tubule suspension, cooling it rapidly to 0 °, and centrifuging it at 48,000 g for 8 minutes. The medium is removed, and the cell pellet is washed twice with ice-cold physiological saline solution. The sides of the tube and the surface of the pellet are dried by suction. The 15M. B. Burg and J. Orloff, Amer. J. Physiol. 217, 1064 (1969). 16R. E. Hillman, I. Albrecht, and L. E. Rosenberg, ]. Biol. Chem. 243, 5566 (1968).
66 4
ISOLATION AND CULTURE OF CELLS
[66]
renal tubules are lysed by adding 2 ml of distilled water and boiling for 10 minutes. Amino acids are determined on the clear supernatant. Kinetic Studies
The above procedure can be used for determination of the steady-state ionic and water content, or of the steady-state accumulation of sugar, amino acid, or p-aminohippurate, but has to be modified for kinetic studies. For 24Na and 42K flux studies, Burg e t al. 17 have proposed a faster sampling procedure. Rapid separation of tubules from the medium can be achieved by placing 1-4 ml of the medium suspension in a BiJchner funnel (inside diameter 55 mm) fitted with filter paper (Whatman No. 541) and filled with 50 ml of ice-cold Na- and K-free diluent, for instance, trihydroxymethylaminomethane (Tris) 155M or sucrose, 310M. Suction is immediately applied, and the filter paper is washed twice with 25 ml of ice-cold Na-, K-flee diluent. Although the procedure requires approximately 15 seconds, Burg e t al. 17 suggest that, at least for ~4Na and 42K, exchange of isotope between the medium and the tissue appears to be terminated immediately on addition of the suspension to the diluent. Filter papers containing the tissue are extracted overnight with 10 ml of 0.75 N.HNO3. Radioactivity is assayed in 1 ml of this extract, and ionic content is measured on the same extract by atomic absorption photometry. To estimate the amount of radioactivity and ions trapped in the filter paper, an amount of radioactive medium equal in volume to the amount of suspension is introduced into the Biichner funnel. The filter paper is washed, extracted, and analyzed for radioactivity and ionic content in the same manner as for the suspension. The amount of ions and radioactivity found trapped in the filter paper is subtracted from the experimental values. These corrections are usually lower than 5 % for Na and less than 1% for K, and thus do not have an important effect on the final results. The amount of cations recovered from tissues after filtration is approximately 60% of the original content of the tissue in an identical volume of suspension. Burg e t al., 1~ however, have well established the fact that the fraction of ions recovered from the filter paper is representative of that present in the tissue. The ratio Na:K is the same in extract of tissue removed by filtration or by centrifugation.1~ Presumably, the loss of cations during filtration could be related to the passage of cells through or around the filter or to cell damage during handling. Murthy and Foulkes TM have used a quite similar procedure to study the movements of PAH labeled with '4C. i'M. B. Burg, E. F. Grollman, and J. Orloff, Amer. J. Physiol. 206, 483 (1964). "SL. Murthy and E. C. Foulkes, Nature (London) 213, 180 (1967).