SPECIAL COMMUNICATION
A simplified method for isolation of large numbers of defined
nephron segments
J. A.
Schafer1,
M. L.
Watkins1,
L.
Li1,
P.
Herter2,
S.
Haxelmans3, and
E.
Schlatter3
1 Department of Physiology and
Biophysics, University of Alabama at Birmingham, Birmingham,
Alabama 35294-0005; 2 Max-Planck
Institut für Molekulare Physiologie, 44026 Dortmund; and
3 Westfälische-Wilhelms-Universität
Münster, Medizinische Poliklinik, Experimentelle Nephrologie,
48149 Münster, Germany
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ABSTRACT |
We describe a
simplified method for the isolation of large numbers of nephron
segments from rat and rabbit kidneys. In contrast to most previous
protocols, the kidneys are not perfused. After removal from the animal,
the kidney is sliced and torn in pieces that are subsequently digested
in culture medium containing 0.5 mg/ml of collagenase at 37°C. If
the preparation is agitated only very gently and infrequently, then the
tissue gradually falls apart into a suspension containing long nephron
fragments, often consisting of multiple connected segments. These are
easily sorted into homogeneous segment populations that can be used for
enzyme assays, protein extraction for immunoblotting, and RNA
extraction for reverse transcription-polymerase chain reaction, all of
which have been done successfully in our laboratory. For comparison, we
have also examined cortical collecting tubule segments and cells
prepared by the more rigorous protocol described previously (E. Schlatter, U. Fröbe, and R. Greger.
Pflügers Arch. 421: 381-387, 1992). Even after the isolation of single cells in a Ca2+-free medium, the cells
maintain their normal architecture and a distinct separation of apical
and basolateral membranes.
microdissection; collagenase; protease; enzyme assays; immunoblotting; scanning electron microscopy; cortical collecting
tubule; rat; rabbit; epithelial transport
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INTRODUCTION |
ISOLATED NONPERFUSED nephron segments have proven to be
an excellent preparation for physiological studies of hormone action and intracellular second messenger systems, as well as for patch-clamp studies of individual membrane surfaces. The use of isolated nephron segments began with studies of phenol red accumulation in
microdissected chick mesonephros by Chambers and Kempton (8) and was
extended by Cameron and Chambers (7) to functional studies in nephron segments from a human embryo. The technique was vastly improved by Burg
and Orloff (6) in their pioneering study, which was the first to
demonstrate the metabolic and transport stability of preparations of
nephron segments from the collagenase-treated rat kidney.
Burg et al. (5) subsequently developed the technique for
microdissecting and perfusing isolated nephron segments without collagenase treatment. Work by this and other laboratories concentrated on this preparation because of the advantages of the in vitro technique
for transepithelial transport processes in regions of the nephron that
could not be accessed directly by micropuncture. However, the isolated
nonperfused tubule preparation was found to be preferable for many
studies of segmental metabolism as refined by Guder et al. (11) and of
receptor localization and intracellular second messenger coupling as
refined by Morel and colleagues (15, 16, 19, 20). For example, using
collagenase treatment, the latter group was able to identify and
isolate the multiple segments of the distal convoluted tubule from the
rabbit (15) and rat (16) and measure their adenosine
3',5'-cyclic monophosphate (cAMP) production in response to
arginine vasopressin (AVP), parathyroid hormone, isoproterenol, and
calcitonin. In the studies in the rabbit, they were able to dissect
entire complexes of multiple distal convoluted tubules, with arcades
and cortical collecting duct (CCD) intact (see, for example, the
micrograph in figure 4 of Ref. 20). The primary shortcoming of this
approach is the skill and labor that is required to dissect the nephron
segments, which results in a practical limitation to quantities that
can be obtained.
Schlatter et al. (25) have recently modified the enzyme incubation
procedure to prepare isolated CCD segments or cell clusters for patch
clamping. In their preparation, the kidneys are perfused in situ with a
medium containing both collagenase and another protease. Slices from
the kidneys are then incubated with the same enzymes at 37°C,
resulting in a more thorough dispersion of the nephron segments. When
the digested slices are subsequently transferred to the dissection
dish, it is found that many individual segments are completely
dissociated from the tissue slices, so that little or no dissection is
required. Thus numerous CCD segments could be gathered merely by
sorting them from other segments. Cell-attached or excised membrane
patch-clamp recordings could then be made from the basal side of the
epithelium of the selected segments. Further treatment of these
segments in a Ca2+-free buffer
produced clusters of highly uncoupled cells that could be used for fast
or slow whole cell patch clamping (25). Despite the enzymatic treatment
and disaggregation, these cells were subsequently shown to have normal
electrophysiological responses to AVP (25, 28) and normal responses of
intracellular Ca2+ and
Na+/H+
exchange to a variety of hormones (1-3, 24, 26).
We undertook the present study to examine the morphological integrity
of the cells isolated by this procedure and to refine the method so
that it would be suitable for the dissection of even larger amounts of
individual nephron segments for RNA extraction for reverse
transcription-polymerase chain reaction (RT-PCR), for multiple
enzymatic assays, and for extraction of sufficient protein for
immunoblotting. Although we (31) and others (e.g., 30) have conducted
RT-PCR on RNA extracted from 1-2 mm of isolated nephron segments,
it is desirable to have more tissue for the detection of low-abundance
messages. McDonough et al. (18) have also microdissected sufficient
amounts of CCD by the conventional collagenase treatment method for
immunoblotting of Na-K-adenosinetriphosphatase isoform proteins;
however, on the order of 100 mm of CCD is required to obtain sufficient
protein (~10 µg) for a single lane on the blot. The method we
describe below allows a single investigator to sort, measure, and
transfer samples of over 50 mm of CCD and other nephron segments within
1 h following digestion. Furthermore, the gentle enzymatic and
mechanical treatment used in this method preserves long segments of the
nephron consisting of multiple regions. This procedure allows for
easier identification and isolation of the desired nephron segments and
decreases the time that must be expended in collecting and transferring
the segments. We have also examined the nephron segments obtained by
previous procedure of Schlatter et al. (25) with scanning electron
microscopy (SEM) and have shown that, even with the more vigorous
enzymatic and Ca2+-free treatment,
the CCD segments and cells maintain their normal morphology and
polarization of the apical and basolateral membranes.
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METHODS |
First, we briefly describe the previously reported method (25) for the
preparation of CCD segments using perfusion of the kidneys and their
further digestion with collagenase plus protease, followed by the
methods used to examine these cells with SEM. Second, we describe our
newly modified procedure, which involves only collagenase digestion
without prior perfusion of the kidneys and which permits dissection of
larger numbers of relatively long nephron segments.
Collagenase plus protease perfusion and digestion
protocol. Isolation of CCD tubule segments and
uncoupled cells according to this protocol was similar to the method
described before (25), and we will refer to it subsequently as
collagenase + protease digestion. For isolation of other tubular
segments such as proximal tubules, thick ascending limbs, or inner
medullary collecting ducts, the enzyme concentrations and incubation
times may require minor modifications. Female Wistar rats (Charles
River, Sulzfeld, Germany) weighing 60-150 g were anesthetized with
intraperitoneal injections of 100 mg/kg body wt Trapanal (Byk-Gulden,
Constance, Germany). The anesthetized rats were placed on a heated
table, and the left kidney was exposed. The renal artery was cannulated with fine polyethylene tubing, and 3 ml sterile culture medium (Ham's
F12; GIBCO, Berlin, Germany) containing 1 mg/ml collagenase (type IV;
Sigma, Deisenhofen, Germany) and 1 mg/ml protease (type XXV, Pronase E,
Sigma) were infused through the kidney within 1 min. The kidney was
removed immediately thereafter, decapsulated, and rinsed in sterile
Ham's F12 medium. Small pieces (~2-mm cubes) were cut from the
cortex. These pieces were incubated at 37°C in 2 ml sterile Ham's
F12 medium containing 0.5 mg/ml collagenase and 0.5 mg/ml protease and
were gassed with 5% CO2-95% air
in a glass vial for ~10 min with mild shaking. At this point the medium contained isolated glomeruli and short tubule segments. After
centrifugation at 3,000 rpm for 2 min, the enzyme-containing solution
was removed, and tubules were resuspended in ice-cold Ham's F12
medium. Individual tubule segments could be identified easily in a
dissection microscope at ×25-40 magnification by their appearance and dimensions. These tubule segments are suitable for
patch-clamp studies (cell attached, excised, and slow or fast whole
cell configuration) with seals obtained from the basal side. The cells
of these tubular segments can also be electrically and physically
separated by an incubation in ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA)-buffered (5 mM)
Ca2+-free medium on ice with
gentle shaking for an additional 10-20 min, which produces cell
clusters with rounded, electrically uncoupled cells (see Fig. 2). This
Ca2+-free incubation resulted in
isolated cells (see Fig. 3) within 10-20 min.
Scanning electron microscopy. Tubules
or cells prepared by the collagenase + protease protocol were seeded on
Thermanox plates (10.5 × 22 mm; Nunc, Naperville, IL) coated with
poly-L-lysine as an adhesive.
Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline
for 1 h and then dehydrated in ethanol solutions with ascending ethanol
content. Thereafter the critical point dried cells were coated with a
10-nm thick layer of platinum and examined using a Hitachi S-800
scanning electron microscope.
Simple collagenase digestion protocol.
The animals used in these studies were male Sprague-Dawley rats (Harlan
Sprague Dawley, Indianapolis, IN) and female New Zealand White rabbits
(Myrtle's Rabbitry, Thompson Station, TN). Rats and rabbits were
obtained 5-14 days before use and were maintained ad libitum on
tap water and standard pelleted rat chow (16% rodent diet no. 8746;
Teklad, Madison, WI) or standard pelleted rabbit chow (Teklad 15%
rabbit diet 8630). At the time the rats were used for an experiment, they weighed 100-170 g, and the rabbits weighed 2-2.5 kg. To
obtain a kidney for isolation of nephron segments, rats were
decapitated, and rabbits were anesthetized with a combination of
ketamine (40 mg/kg) and xylazine (10 mg/kg) and decapitated when
surgical anesthesia had been achieved.
In both species the kidneys were rapidly harvested after decapitation,
and they were not perfused with any solution before removal. The
capsules were stripped away, and 0.5- to 1.0-mm thick slices of tissue
were made using a Thomas-Stadie-Riggs tissue slicer (Thomas Scientific,
Swedeboro, NJ). When we wished to dissect nephron segments from the
cortex, the slices were cut tangential to the cortical surface opposite
the hilum. For the rat kidney, only one cortical section was taken,
because the thickness of the cortex in rats of this size is only
slightly more than thickness of the slice. For the rabbit kidney, which
has a cortical thickness of ~4 mm, at least three slices could be
taken. The sections from both species were carefully examined to be
sure they contained no medullary tissue. Coronal slices were used to
dissect nephron segments from the medulla or from medullary rays. In
the first case, the cortical tissue was removed before the tissue was
digested, and in the latter case, the medullary tissue was removed.
Slices of cortex or medulla were then torn into small chunks using
forceps (two to four pieces in the case of the rat slices and twice as
many for the rabbit slices). These pieces of tissue were incubated in 2 ml of warm Eagle's minimal essential medium (MEM)
containing 0.5 mg/ml of type 2 collagenase, 5 mM glycine, 50 U/ml deoxyribonuclease (DNase), and 48 µg/ml of soybean
trypsin inhibitor in a 20-ml scintillation vial. [All reagents
were from Sigma Chemical, St. Louis, MO, with the exception of the
collagenase (catalog no. 4176), which was obtained from Worthington
Biochemical, Freehold, NJ.] We refer to this medium below as
"MEM-collagenase mixture." The cortical pieces were briefly (<5
s) and very gently agitated by hand and incubated without shaking at
37°C while exposed to room air. (We have also used a standard
tissue culture incubator gassed with air plus 4%
CO2 for the procedure, but this
does not seem to be necessary.) At ~15-min intervals, the
supernatant was poured off the sedimented tissue into a 5-ml ice-cold
test tube. Fresh MEM-collagenase medium was added to the remaining
tissue pieces, and digestion could be continued with supernatant
removal at 10- to 15-min intervals in the same manner until the large sections were exhausted. Cloudy, tubule-rich supernatants were evident
after 30-40 min of digestion. The progress of the digestion was
followed by examining droplets of the supernatant under a dissecting
microscope.
It was found that tubule segments sedimented rapidly in the test tubes.
The supernatant overlying the tubule segments was carefully removed
with a Pasteur pipette and replaced with 2 ml of ice-cold, enzyme-free
dissection solution containing 1% bovine serum albumin (BSA), and the
tubules were stored on ice until sorting began. We expected that the
BSA would bind residual collagenase and any contaminating proteases,
and this would help to prevent further digestion.
For tubule selection, aliquots of the tubule-rich suspension were
gently pipetted, using a large bore transfer pipette, into a dissection
dish containing MEM (we have also used Ham's F12 medium successfully)
with 0.05% BSA at 4°C. Tubule segments were sorted from the
central group of tubule segments by using a 30-gauge needle to move
them to a clean area of the dish. Selection of tubule segments could be
carried out over 3-4 h. The recovered segments were measured
individually with an ocular micrometer, and groups of selected segments
with a total length of 20 mm were transferred in 10 µl of medium with
a P-20 Pipetman (Eppendorff Scientific, Madison, WI) as needed for
further experimentation.
To examine the viability of the nephron segments, several
"control" tubules were transferred in a 10-µl sample to a
10-µl drop of MEM containing 0.4 g/dl of trypan blue. If more than
five stained cells were found in a 0.5-mm length, none of the samples was used. When the tubule segments were to be used for RNA or protein
extraction, they were rinsed by transferring them to a 1.5-ml
microcentrifuge tube containing ice-cold fresh medium. The tubules were
briefly centrifuged at <1,000 rpm, and the supernatant was removed by
pipette. The RNA extraction medium (TRIzol; Life Technologies,
Gaithersburg, MD) or the protein extraction buffer was immediately
added to the pellet, which dissolved instantaneously.
For the purposes of the present methodological study, we pipetted a
50-µl droplet containing the tubules onto a microscope slide and
covered the droplet with a large coverslip. The tubules were examined
using a Nikon Diaphot inverted microscope equipped with ×10
wide-field eyepieces. Incident light was either bright-field diffused
through a white frosted filter or passed through a Hoffman condenser
(Modulation Optics, Greenvale, NY). The objectives were as follows: a
Nikon ×2 Plan, a Nikon ×10 Plan modified by Modulation Optics for Hoffman modulation contrast microscopy, and a Nikon ×60/1.40 Plan Achromat oil-immersion lens. In the case of these light micrographs (Figs. 1-4),
photographs were taken using 35-mm Kodak T-Max 100 black and white
film. Images were digitized from the negatives using a Management
Graphics Solitaire 8xp Film Recorder (Dept. of Photography and
Instructional Graphics, Univ. of Alabama). The images were digitally
cropped, sized, and labeled, the contrast was optimized by Photoshop
software (Adobe Systems, Mountain View, CA), and the image files were
prepared for electronic transmission directly to the publisher (via
FTP). Intermediate hard copies were produced using an Epson Color
Stylus II ink jet printer.

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Fig. 1.
Scanning electron microscopy (SEM) of an enzymatically isolated rat
cortical collecting duct (CCD) segment obtained according to the
collagenase + protease protocol without
Ca2+-free treatment.
Magnification, ×1,000. Dotted line = 30 µm.
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RESULTS AND DISCUSSION |
Collagenase + protease perfusion and digestion
protocol. The protocol was used only to prepare cells
for examination by SEM and thereby to verify that even the more
vigorous enzymatic and Ca2+-free
treatments did not alter the cells structurally. Figure 1 shows a rat
CCD segment near a bifurcation. Segments such as these were observed
after collagenase + protease treatment but without
Ca2+-free treatment. Figure
2 shows a cluster of rat CCD cells prepared according to the collagenase + protease protocol followed by 15 min of
Ca2+-free incubation on ice. The
isolated CCD cell shown in Fig. 3 was also
prepared by the collagenase + protease protocol followed by 15 min of
Ca2+-free incubation. In all
cases, it can be seen that there is excellent preservation of the fine
structure of the cells. More importantly, even when the cells are
completely isolated, as in Fig. 3, they maintain their polarity.

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Fig. 2.
SEM of a cluster of rat CCD cells obtained according to the collagenase + protease protocol followed by a 15-min incubation in
Ca2+-free, EGTA-buffered medium.
Note the differences in the surface structures of individual cells.
Magnification, ×5,000. Dotted line = 6 µm.
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Fig. 3.
SEM of a single rat CCD cell obtained according to the collagenase + protease protocol following a 15-min incubation in a
Ca2+-free, EGTA-buffered medium.
Note the differences in the surface structures of the cell with the
basal infoldings (bottom left) and
microvilli-like structures (top
right). Magnification, ×10,000. Dotted line = 3 µm.
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The CCD segments obtained by the collagenase + protease procedure have
been used for several physiological experiments including microfluorometric determination of cellular
Ca2+ activities (2, 23, 26),
cellular Na+ activities (23),
cellular pH (25, 27), and patch-clamp experiments to record membrane
voltages (14, 25, 27, 28), whole cell conductances (25, 27), or single-
channel currents (3, 12, 13, 14, 25, 27). The enzymatic
isolation procedure including the
Ca2+-free incubation apparently
does not remove or inactivate hormone receptors, because in several
studies we could functionally demonstrate the existence of
V1 and
V2 receptors (2, 25, 26), of
acetylcholine receptors (2), of purinoceptors and angiotensin II
receptors (26), and of
-receptors (2).
The same methods have been used previously to obtain medullary and
cortical thick ascending limbs (MTAL and CTAL) and inner medullary
collecting duct segments. Viability of the segments was indicated by
intracellular voltage and pH measurements made in the case of the
ascending limb segments (4) and by hyperpolarization of the
intracellular voltage upon amiloride addition in the case of the inner
medullary collecting ducts (unpublished observations).
Simple collagenase digestion protocol.
Figures 4 and 5
show samples of nephron segments isolated from, respectively, rat and rabbit kidneys treated by the collagenase digestion protocol. As shown
in Fig. 4A, the supernatant drawn from
the collagenase digestion vial and placed in the dissection dish
consisted of numerous separated nephron segments. Using either a
30-gauge needle or a fine forceps (Dumont no. 5), we found it quite
easy to separate like tubule segments into collections in a clean area
of the dissection dish from which they were subsequently transferred
for microscopic observation or for experiments. In the case of cortical
tissue slices, complexes of connected CCD, connecting tubules (CNT), and distal tubules (DT) were observed frequently and could easily be
trimmed to isolate individual segments. For example, we frequently observed preservation of the entire structure of the DT from the CTAL
of the loop of Henle to the CCD (e.g., see Fig.
4G), which allowed easy
identification of the macula densa region, early (bright) DT, late
(granular) DT, and the CNT segments. We could also obtain full lengths
of all the medullary segments from the coronal kidney slices (Fig. 4,
C-E).

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Fig. 4.
Rat nephron segments prepared by collagenase protocol.
A: tubule fragments immediately after
preparation and as they appear in dissection dish before sorting.
B: a proximal tubule segment extending
from early proximal convoluted region to early proximal straight
segment (bottom right).
C: a segment containing the proximal
straight tubule (left) and thin
descending limb of the loop of Henle.
D: thin ascending limb
(left) extending through the
medullary (MTAL) to the beginning of the cortical thick ascending limb
(CTAL, right).
E: MTAL of the loop of Henle
(top) and thin ascending limb of the
loop of Henle (bottom).
F: CCD extending to the
bottom right is formed from the
confluence of two connecting tubules (CNT). Branch on
left extends to a distal convoluted
tubule with a glomerulus still attached at the region of the macula
densa and to the CTAL. G and
H: two additional examples of CCDs
formed from the confluence of multiple connecting and distal tubule
(DT) segments. Micrograph in A was
taken with bright-field illumination and the ×2 lens; all other
micrographs were taken with the ×10 lens and Hoffman contrast
modulation. Nephron segments shown in
A, B,
and
F-H
came from cortical tissue slices tangential to the cortical surface,
whereas those in
C-E
came from coronal slices of the medulla.
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Fig. 5.
Rabbit cortical nephron segments prepared by collagenase protocol.
A: collection of nephron segments
sorted from the dissection dish and transferred to a microscope slide
in a droplet of MEM. B: a CCD
(right) with attached CNT and DT
segments. C: proximal convoluted
tubule. D: Short segment of CCD
(pointing toward the bottom left)
and attached connecting and DT segments. Micrograph in
A was taken with bright-field
illumination and the ×2 lens; all other micrographs were taken
with the ×10 lens and Hoffman contrast modulation.
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We found the most critical aspect of the collagenase digestion
procedure and the subsequent transfer of the nephron segments was to
avoid mechanical stresses that would shear the tubule segments. When
the suspensions were aerated by bubbling, when the tubules were
resuspended by swirling, or when the tubules were centrifuged even
briefly at low speed, the segments were uniformly found to be much
shorter. Therefore, when the tubules were to be rinsed with fresh
medium, they were allowed to settle to the bottom of a container such
as a microcentrifuge tube, the old medium was pipetted from the top,
and the new medium was slowly introduced down the side of the
container. When tubule segments were later transferred from the
dissection dish to a microscope slide or to another container for
further processing, groups of 5-10 were drawn slowly into a
20-µl Eppendorf Pipetman tip and slowly ejected, with both procedures
being conducted under observation with the dissecting microscope so
that a complete transfer of the measured length could be confirmed. In
this way a single investigator could separate, measure, and transfer
over 50 mm of a given nephron segment within 1 h.
The integrity of the isolated segments was examined by testing their
ability to exclude the vital dye trypan blue, which has been a frequently used criterion for viability (9). Figure 6 shows an example of a CCD-DT complex from
a rat kidney that was purposely damaged by incubation at room
temperature. Several cells that stained with trypan blue can be seen
with the ×10 objective, and with the ×60 objective it can
be seen that even those cells that are not stained with trypan blue are
swollen and granular. Using trypan blue and cell appearance at high
power as indicators of damage, we found that the tubules remained
viable for at least 3 h when kept either at 37°C in an incubator
gassed with air plus 4% CO2 or at
4°C (as in the dissection dish). On the other hand, even relatively
short incubation (<30 min) at room temperature with or without
gassing caused significant cell damage, as seen in Fig. 6. We also
varied the concentration of collagenase in the MEM-collagenase mixture
and found that we could reduce it to 0.5 mg/ml and maintain good tissue
digestion. This concentration is considerably lower than the 2 mg/ml
more typically used in previous procedures.

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Fig. 6.
Effect of incubation at room temperature on tubule integrity.
A: complex of rat CCD, CNT, and DT
segments (Hoffman contrast modulation ×10 objective) was
incubated at room temperature for 40 min in MEM medium and then
transferred in 10 µl of the medium to an equal volume of medium
containing 0.4% trypan blue. Damaged cells that are permeable to the
vital dye can be identified by the dark spots corresponding to nuclei.
B: (×60 oil-immersion objective)
higher magnification of the region of the leftmost confluence of two
distal segments in A shows four cells
with nuclei that are stained by trypan blue. Other cells are swollen
and granular in appearance.
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It is not clear why the tubules are more labile at room temperature,
although we might speculate that, because metabolic and transport
processes have differing temperature sensitivities, the persistence of
some metabolism at room temperature causes greater cell damage than the
cessation of all metabolism at 4°C. Also, it has long been standard
procedure to use ice-cold solutions for tissue preservation, for
example, in the preservation of organs for transplantation. In this
regard, it might be possible to preserve the nephron segments from the
digested kidney slices even longer if we were to use a hyperosmotic
solution with a composition more like that of intracellular fluid (21,
22) as is common practice in organ transplantation. However, we did not
find this necessary in these experiments.
We have done several series of experiments in which RNA was extracted
from isolated proximal tubule and CCD segments for amplification by
RT-PCR (29, 32), and the method provides more than enough RNA for the
detection of even low-abundance messages. We have also used this method
for analysis of adenylyl cyclase activity in isolated, nonpermeabilized
rat CCD segments. These studies confirmed markedly enhanced cAMP
production with 20 or 200 pM AVP and that this stimulation could be
inhibited by either 100 nM epinephrine or 10 µM dopamine (17). These
observations demonstrated that receptors for all three hormones were
intact and that metabolic ATP production and adenylyl cyclase activity
were normal. This was expected because, even with the more vigorous
isolation using the collagenase + protease protocol followed by
disaggregation of the cells in a
Ca2+-free buffer, there was normal
receptor and transport activity as discussed above.
The method is also valuable for extracting sufficient protein from a
discrete nephron segment for immunoblotting. We have found that
10
µg of protein can be isolated from 100 mm of proximal and/or
CCD segments and that this is sufficient for immunoblotting of all but
very low-copy proteins. This can be compared with the observations of
McDonough et al. (18), who found that 6.8 µg of protein could be
extracted from 40 mm of proximal straight tubule but that only 0.12 µg of protein could be extracted from 40 mm of CTAL or MTAL. On the
other hand, 100 mm of proximal or CCD segments provides only on the
order of 10 ng of total RNA after standard preparation by guanidium
isothiocyanate/acid phenol extraction with one propanol and one ethanol
precipitation. This small amount of total RNA is, unfortunately,
insufficient for the isolation of mRNA for either Northern blotting or
ribonuclease protection assays. Thus other methods, such as
immunodissection (10), will have to be perfected to obtain larger
amounts of RNA from a pure segment population.
Nevertheless, this method provides a simple and quick method for the
preparation of relatively large amounts of individual tubule segments
that are very easily identified and thus homogeneous, and the method
does provide sufficient material for immunoblotting of proteins and for
RT-PCR of mRNA from individual nephron segments. If samples from
multiple investigators were pooled, then it would be possible to obtain
sufficient RNA to prepare cDNA libraries of specific nephron segments
from rats and rabbits that have been maintained and monitored while on
a variety of dietary protocols.
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ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-25519 and DK-45768, by Deutsche
Forschungsgemeinschaft Schl 277/2-3 to 2-5, and
by a grant from the Max-Planck-Society and the von Humboldt Foundation.
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FOOTNOTES |
Address for reprint requests: J. A. Schafer, Univ. of Alabama at
Birmingham, Dept. of Physiology and Biophysics, 1918 Univ. Blvd., Rm.
958, Birmingham, AL 35294-0005.
Received 4 February 1997; accepted in final form 15 May 1997.
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