[ 1 8]
METABOLISM OF ISOLATED TUBULE SEGMENTS
325
It should be mentioned in conclusion that similar binding micromethods can be applied to investigate the action sites of other drugs and certain diuretics which are currently employed for pharmacological studies of kidney functions, sS-s8 Acknowledgments The authors are indebted to Mrs. Violette Biausque and to Mrs. Sylvie Siaume- Perez for their help in preparing the manuscript. a5 W. N. Suki, B. J. Stinebaugh, J. P. Frommer, and G. Eknoyan, in "The Kidney: Physiology and Physiopathology" (D. W. Scldin and G. Giebisch, eds.), Vol. 2, p. 2127. Raven, New York, 1985. a6 E. Giesen-Crouse, P. Frandeleur, M. Schmidt, J. Schwartz, and J. L. Imbs, J. Hypertension 3, $211 0985). a7 p. Barbry, C. Frelin, P. Vigne, E. J. Cragoe, Jr., and M. Lazdunski, Biochem. Biophys. Res. Commun. 135, 25 (1986). as p. Vigne, M. Lazdunski, and C. Frelin, Eur. J. Pharmacol. 160, 295 0989).
[ 18] M e t a b o l i s m o f I s o l a t e d K i d n e y T u b u l e S e g m e n t s 1
By GABRIELE
WIRTHENSOHN and WALTER G. GUDER
Introduction In the last century morphologists had already observed that the kidney consists of different structural and functional units. Each kidney contains about 106 nephrons with giomeruli producing the primary filtrate and the tubule forming the final urine in many subsequent segments. To study the physiological and biochemical functions of the different nephron segments (Fig. 1) special techniques have been developed. Micropuncture and microperfusion were used to study tubular transport. Biochemical functions (enzymes, substrates, metabolic pathways) were studied mainly in whole kidney homogenates and tissue slices until the late 1950s, when more special techniques were developed which allowed biochemical studies at the defined segment level. In this chapter we present a short review on the isolation and analysis of defined nephron segments, giving some examples of the determination of enzyme activities and metabolic pathways in them. The advantages and disadvantages of the different techniques are also discussed. In memory of Helen B. Burch.
METHODS IN ENZYMOLOGY,VOL. 191
Copyright© 1990by AcademicP r ~ Inc. Allrightsof rt'laroductionin any formreserved.
326
KU~NEY
[18]
FIG. 1. Structural organization of the rat kidney. A, proximal convoluted tubule (PCT); B, proximal straight tubule (pars recta, PR); C, thin descending limb of Henle's loop (TDL); D, medullary thick ascending limb of Henle's loop (MAL); E, cortical thick ascending limb of Henle's loop (CAL); F, distal convoluted tubule (DCT); G, cortical collecting tubule (CCT); H, medullary collecting tubule (MCT).
Isolation Procedures for Defined Segments Tubule Suspensions Cortical Tubules. In 1962, Burg a n d Orloff ~a isolated tubule suspensions f r o m rabbit renal cortex with the aid o f collagenase. T h e suspensions exhibited morphologic a n d metabolic properties o f proximal tubules. This m e t h o d was later extended to rat, 2-4 dog, 5 mouse, 6 a n d h u m a n 7 kidney. This preparation was morphologically d e m o n s t r a t e d to contain m o r e t h a n 90% proximal tubules. 8 An additional Percoll gradient step allows further
~aM. B. Burg and J. Orloff, Am. J. Physiol. 203, 327 (1962). 2N. Nagata and H. Rasmussen, Proc. Natl. Acad. Sci. U.S.A. 65, 368 (1970). 3 W. G. Guder, W. Wiesner, B. Stukowski, and O. Wieland, Hoppe-Seyler's Z. Physiol. Chem. 352, 1319 (1971). + W. G. Guder, Biochim. Biophys. Acta 584, 507 (1979). 5G. Baverel, M. Bonnard, E.d'Armagnac de Castanet, and M. Pellet, Kidney Int. 14, 567 (1978). 6 G. Wirthensohn and W. G. Guder, Pfluegers Arch. 404, 94 (1985). 7 G. Baverel, M. Bonnard, and M. Pellet, FEBS Lett. 101, 282 (1979). s W. Pfaller, W. G. Guder, G. Gstraunthaler, P. Kotanko, J. Jehart, and S. Pfirsehel, Biochim. Biophys. Acta 805, 152 (1984).
[ 18]
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purification9 and separation of a small fraction of pure distal convoluted tubule segments.~° This preparation can be applied to all metabolic studies of function which have been found to be preferentially, if not exclusively, localized in the proximal tubule. Renal gluconeogenesis, amino acid, lipid, and ketone body metabolism, as well as oxygen consumption and hormonal regulation and degradation exhibited optimal rates when studied in this model.H In addition this preparation can be used to study intracellular compartmentat i o n $'12 as well as membrane composition of proximal tubules? 3 Medullary Tubules. More recently a similar technique has been applied to prepare tubules from the outer and inner medulla? 4,~5 Outer medullary tubule suspensions consist mainly of thick ascending limb segments, contaminated with other tubule types and cellular debris which can be removed by density gradient centrifugation on Percoll leading to a suspension of 96% thick ascending limbs.14 When studying oxygen consumption, substrate metabolism, and hormone action, they establish properties of the mitochondria-rich cells of the ascending limb of Henle's loop. Inner medullary tubule suspensions consist mainly of collecting tubule segments with some thin limbs and can be used as a model for studying metabolism of the renal inner medulla. ~5
Isolated Cells With the aid of calcium chelators [ethylenediaminetetraacetic acid (EDTA), citrate], tight junctions connecting tubule cells can be weakened to obtain isolated cell suspensions. These were prepared from rabbit renal cortex 16 and outer medulla 17 and further purified by free-flow electrophoresis. ~sThis procedure led to proximal and distal tubule cells of high purity, whose metabolic functions, however, were not so well maintained compared to tubule suspensions. ~9 Further purification of distal cell types can 9 D. W. Scholer and I. S. Edelman, Am. J. Physiol. 237, F350 (1979). ~op. Vinay, A. Gougoux, and G. Lemieux, Am. J. Physiol. 241, F403 (1981). H G. Wirthensohn and W. G. Guder, Physiol. Rev. 66, 469 (1986). ~2W. G. Guder and S. POxsehel, Int. J. Biochem. 12, 63 (1980). 13C. LeGrimellec, M.-C. Giocondi, B. Carriere, S. Carriere, and J. Cardinal, Am. J. Physiol. 242, F246 (1982). t4 M. E. Chambedin, A. LeFurgey, and L. J. Mandel, Am. J. Physiol. 247, F955 0985). ~5G. Wirthensohn, F. X. Beck, and W. G. Guder, PfluegersArch. 4109,411 (1987). ~6p. Poujeol and A. VandewaUe,Am. J. Physiol. 249, F74 (1985). ~7j. Eveloff, W. Haase, and R. Kinne, J. CellBiol. 87, 672 (1980). n A. Vandewalle, B. Ktpfer-Hobelsberger, and H.-G. Heidrich, J. Cell Biol. 92, 505 (1982). ~9H.-G. Heiddch, A. Vandewalle, B. Ktpfer-Hobelsberger, and W. G. Guder, in "Cell Function and Differentiation" (A. Evangelopoulos, ed.). Liss, New York, 1982.
328
KIDNEY
[18]
be obtained by immunoabsorption and lectin absorption techniques2°-22 and can be used as a basis for metabolic studies and cell culture techniques.
Microdissection Procedures At the beginning of this century methods were developed to isolate single nephrons.23 This maceration technique was done by treatment of the tissue with 75% hydrochloric acid. Although proteins were denatured, well-preserved epithelial structures could be obtained. To isolate discrete nephron segments for the study of enzyme activities or metabolic pathways, however, two different techniques have been applied and have become well established in the past 20 years. Microdissection from L yophilized Tissue Sections. This technique, initially developed by Linderstrom-Lang et al.U for metabolic studies in microbial cells, was extended and amended by O. Lowry and associates25 for microdissection of freeze-dried tissue sections. This technique is well suited to the isolation and identification of single tubular structures from freeze-dried renal sections.26 In this procedure fresh kidney tissue is quickly cut into small cones of 2-mm thickness, mounted on a tissue holder, and plunged into liquid nitrogen to maintain structural and biochemical integrity and reduce the development of ice crystals. 27 The tissue sample is transferred to a cryostat and sections of 16-#m thickness are cut with a microtome. This procedure preserves the fuli activity of most enzymes (for exceptions, see Pros and Cons of the Microdissection Procedures, below). Single segments are dissected using an alternate periodic acid-Schiff base-stained section as a guide.26 Single tubular structures obtained from these tissue sections are weighed on a quartz fiber balance calibrated with quinine crystals. 26 Dry weights of about 1 - 5 0 ng can be quantified with this method. 2oM. Le Hir and U. C. Dubach, Histochemistry 74, 521 (1982). 21 W. S. Spielman, W. K. Sonnenberg, M. L. Allen, L. J. Arend, K. Gerozissis, and W. L. Smith, Am. J. Physiol. 251, F348 (1986). 22A. Vandewalle, M. Tauc, F. Cluzeaud, P. Ronco, F. Chatelet, P. Verroust, and P. Poujeol, Am. J. Physiol. 250, F386 (1986). 23K. Peter, "Untersuchungen tiber Bau und Entwicldung der Niere." Gustav Fischer Verla~ Jena, German Democratic Republic, 1909. u K. Linderstrom-Lang, H. Holter and A. S. Ohlson, C.R. Lab. Carlsberg, Ser. Chim. 21,315 (1938). 22O. H. Lowry, J. Histochem. Cytochem. 1, 420 (1953). 26U. Schmidt and U. C. Dubach, Prog. Histochem. Cytochem. 2, 185 (1971). 27U. Schmidt, U. C. Dubach, W. G. Guder, B. Funk, and K. Pads, in "Biochemical Aspects of Kidney Function" (S. Angielsld and U. C. Dubach, eds.), p. 22. Huber, Bern, Switzerland, 1975.
[ 18]
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The amount of tissue needed depends on the sensitivity of the assay procedure and the activity/concentration of the measured enzyme/substrate in the respective segment. Microdissection from Fresh Tissue. This method, first described by Burg and Odoff in 1962,~" is based on the free-hand preparation of single nephron segments from fresh renal tissue. The procedure is, however, limited to kidneys of animals with easily accessible connective tissue, as is found in rabbit kidney. To separate different medullary and distal nephron structures in other animals, collagenase pretreatment of the kidney is needed. This disintegration of renal connective tissue by collagenase was extensively examined by Morel and co-workers2s and has been applied by many different groups29-33 to overcome the problem of intranephron heterogeneity. For the preparation of single-tubule samples, kidneys are perfused with isotonic saline supplemented with glucose, bovine serum albumin, and collagenase. 28,~,35 After ligation of the renal vein and the ureter the same solution is injected under pressure until rupture of the renal capsule. The kidney is sliced along the corticomedullary axis and small pyramids are incubated at 35-37* for 20-40 min (depending on the animal) in the same collagenase buffer bubbled with a stream of air. The small tissue pyramids are washed with collagenase-free buffer and microdissection is carried out with fine needles under a stereomicroscope. Each dissected nephron segment is photographed for confirmation of identity and measurement of tubular length. Figure 2 shows some representative photographs of tubular segments of rabbit nephron.
Advantages and Limitations of the Various Isolation Procedures Tubule Suspensions. This preparation has the great advantage that many identical samples of relatively well-defined segments can be studied. Routine methods may be applied for metabolite, oxygen, and enzyme activity measurements. Compared to isolated cells and tubule-derived cell 28M. Imbert, D. Chabard~s, M. Monttgut, A. Clique, and F. Morel, Pfluegers Arch. 354, 213 (1975). 29A. Doueet, A. I. Katz, and F. Morel, Am. J. Physiol. 237, FI05 (1979). 30H. Endou, H. Nonoguehi, J. Nakada, Y. Takehara, and H. Yamada, in "Kidney Metabolism and Function" (R. Dzurik, B. Lichardus, and W. G. Guder, ¢kls.) p. 26. Nijhoff, Boston, Massachusetts, 1985. 3~R. M. Edwards, B. A. Jackson, and T. P. Dousa, Am. J. Physiol. 238, F269 (1980). 32N. Farman, A. VandewaUe, and J.-P. Bonvalet, Am. J. Physiol. 244, F325 (1983). 33G. Wirthensohn, A. VandewaUe, and W. G. Guder, Kidney Int. 21, 877 (1982). F. Morel, Am. J. Physiol. 240, F159 (1981). 35A. Vandewalle, G. Wirthensohn, H.-G. Heidrieh, and W. G. Guder, Am. J. Physiol. 240, F492 (1981).
[ 18]
METABOLISM OF ISOLATED TUBULE SEGMENTS
331
cultures a high degree of viability and differentiation is maintained. On the other hand, several limitations have to be considered when using these preparations. Besides effects of collagenase, preparations are always contaminated with minor fractions of other cell types. This can be especially disturbing if isotopes are applied to study metabolic pathways. Likewise minor contaminations can lead to major errors if the studied enzyme is very low in the main cell type.36 In addition, metabolite levels may change during preparation and therefore must be compared with the in vivo levels. Pros and Cons of the Microdissection Procedures. A great advantage of the "Lowry" technique is the low amount of tissue needed for preparation (< 10 mg fresh weight). Moreover, the rapid freezing of the tissue in liquid nitrogen preserves the in vivo conditions. This is of special importance for the study of human tissue, which is available only as biopsy or after nephrectomy. The technique allows storage of tissue in a deep-frozen state over several months to years before dissection is performed. In the hands of a well-trained person this microdissection procedure provides a high degree of flexibility and accuracy. Limitations, on the other hand, arise mainly from the fact that this isolation technique does not provide full integrity of tubular cells. For this reason metabolic pathways and hormone actions cannot be studied. Moreover several enzymes, such as pyruvate carboxylase37 and adenylate cyclase (W. G. Guder, unpublished observations), lose their activity on lyophilization and/or freezing. In contrast to fresh tissue dissection the former method does not allow the dissection of different segments in one tubule, offering the possibility of studying intranephron heterogeneity. Furthermore, only fresh tissue dissection is suitable for studying the sites of hormone actions~,2s,34,38and metabolic pathways such as tdacylglyceroP9 and glucose4° synthesis as well as CO2,41'42 NH 3,43,44 and lactate45 formation.
W. G. Guder and A. Rupprecht, Eur. J. Biochem. 52, 283 (1975). 37 H. B. Butch, R. G. Narins, C. Chu, S. Fagioli, S. Choi, W. McCarthy, and O. H. Lowry, Am. J. Physiol. 235, F246 (1978). 38F. Morel and A. Doucet, Physiol. Rev. 66, 377 (1986). 39W. G. Guder and G. Wirthensohn, in "Biochemistry of Kidney Functions" (F. Morel, ed.), p. 95. Elsevier, Amsterdam, 1982. 40A. Maleque, H. Endou, C. Koseki, and F. Sakai, FEBS Lett. 166, 154 (1980). 41 F. LeBouffant, A. Hus-Citharel, and F. Morel, PfluegersArch. 401, 346 (1984). 42F. LeBouffant, A. Hus-Citharel, and F. Morel, in "Biochemistry of Kidney Functions" (F. Morel, ed.), p. 363. Elsevier, Amsterdam, 1982. 43 H. Nonoguchi, S. Uchida, T. Shiigai, and H. Endou, PfluegersArch. 403, 229 (1985). 44 D. W. Good and M. B. Burg, J. Clin. Invest. 73, 602 (1984). 45S. Bagnasco, D. Good, R. Balaban, and M. Bur~ Am. J. Physiol. 248, F522 (1985).
332
KIDNEY
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The disadvantage of fresh tissue dissection lies in the use of collagenase, which may alter tubular membrane permeabilities and affect enzyme activities. 33,35,46In addition the material is of limited use in the measurement of metabolites since the preparation and incubation procedure may change their concentrations. If regulation in vitro is, however, to be studied at the single-nephron level, freshly dissected tubules are adequate. Microanalytical Procedures To study metabolite concentrations, enzyme activities, hormone actions, or metabolic pathways in single-nephron segments obtained by the microdissection procedures described, sensitive microassays are needed which allow the detection of specific products/metabolites in the picomolar range. This can be achieved by either of the following techniques, which have been successfully applied to renal tissue. Considerable increase in sensitivity can be obtained by decreasing assay volumes to the nanoliter range. This tiny volume is pipetted into small holes (4 × 3 mm) of a Teflon rack sealed with a drop of paraffin oil (oil well) to prevent evaporation.47 Reactions in a few microliters can be performed between two glass slides with opposed depressions, an arrangement allowing radiochemical41,42and cycling3° procedures to be performed. Table I summarizes enzymes, substrates, and pathways which have been successfully analyzed with one of the following techniques.
Enzymatic Cycling Enzymatic cycling analyses were performed with methods based on specific enyzmatic reactions which result in oxidation or reduction of pyridine nucleotides.47 Amplification of signals is obtained by use of enzymatic cycling of nucleotides.48 The following examples illustrate the principles of enzymatic cycling as it is used to study enzyme activities or renal metabolites. Citrate Synthase 49. The tissue to be studied is added to a reagent that carries out the following reaction: Oxaloacetate + acetyl - CoA ---, citrate + CoA 4~ G. Wirthensohn, A. VandewaUe, and W. G. Guder, Biochem. J. 198, 534 (1981). 47 O. H. Lowry and J. V. Passonneau, "A Flexible System of Enzymatic Analysis," Academic Press, New York, 1972. 4s O. H. Lowry, Mol. Cell. Biochem. 32, 135 (1980). 49 H. B. Butch, in "Biochemistry of Kidney FUnctions" (F. Morel, ed.), p. 297. Elsevier, Amsterdam, 1982.
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After oxaloacetate is destroyed by addition of NaOH and heating, a solution is added containing reagents which catalyze the following reaction: Citrate
, acetate + oxaloacetate NADH ~
_malate NAD +
Excess NADH is destroyed by hydrochloric acid and the following cycle is carded out for 60 min: Malate. NAD + _ ethanol
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acetaldehyde
The cycling enzymes are malate dehydrogenase and alcohol dehydrogenase. The cycle is stopped and malate measured in the indicator reaction with the malate dehydrogenase and aspartate aminotransferase reagent: glutamate~_
METABOLISM OF ISOLATED TUBULE SEGMENTS
325
It should be mentioned in conclusion that similar binding micromethods can be applied to investigate the action sites of other drugs and certain diuretics which are currently employed for pharmacological studies of kidney functions, sS-s8 Acknowledgments The authors are indebted to Mrs. Violette Biausque and to Mrs. Sylvie Siaume- Perez for their help in preparing the manuscript. a5 W. N. Suki, B. J. Stinebaugh, J. P. Frommer, and G. Eknoyan, in "The Kidney: Physiology and Physiopathology" (D. W. Scldin and G. Giebisch, eds.), Vol. 2, p. 2127. Raven, New York, 1985. a6 E. Giesen-Crouse, P. Frandeleur, M. Schmidt, J. Schwartz, and J. L. Imbs, J. Hypertension 3, $211 0985). a7 p. Barbry, C. Frelin, P. Vigne, E. J. Cragoe, Jr., and M. Lazdunski, Biochem. Biophys. Res. Commun. 135, 25 (1986). as p. Vigne, M. Lazdunski, and C. Frelin, Eur. J. Pharmacol. 160, 295 0989).
[ 18] M e t a b o l i s m o f I s o l a t e d K i d n e y T u b u l e S e g m e n t s 1
By GABRIELE
WIRTHENSOHN and WALTER G. GUDER
Introduction In the last century morphologists had already observed that the kidney consists of different structural and functional units. Each kidney contains about 106 nephrons with giomeruli producing the primary filtrate and the tubule forming the final urine in many subsequent segments. To study the physiological and biochemical functions of the different nephron segments (Fig. 1) special techniques have been developed. Micropuncture and microperfusion were used to study tubular transport. Biochemical functions (enzymes, substrates, metabolic pathways) were studied mainly in whole kidney homogenates and tissue slices until the late 1950s, when more special techniques were developed which allowed biochemical studies at the defined segment level. In this chapter we present a short review on the isolation and analysis of defined nephron segments, giving some examples of the determination of enzyme activities and metabolic pathways in them. The advantages and disadvantages of the different techniques are also discussed. In memory of Helen B. Burch.
METHODS IN ENZYMOLOGY,VOL. 191
Copyright© 1990by AcademicP r ~ Inc. Allrightsof rt'laroductionin any formreserved.
326
KU~NEY
[18]
FIG. 1. Structural organization of the rat kidney. A, proximal convoluted tubule (PCT); B, proximal straight tubule (pars recta, PR); C, thin descending limb of Henle's loop (TDL); D, medullary thick ascending limb of Henle's loop (MAL); E, cortical thick ascending limb of Henle's loop (CAL); F, distal convoluted tubule (DCT); G, cortical collecting tubule (CCT); H, medullary collecting tubule (MCT).
Isolation Procedures for Defined Segments Tubule Suspensions Cortical Tubules. In 1962, Burg a n d Orloff ~a isolated tubule suspensions f r o m rabbit renal cortex with the aid o f collagenase. T h e suspensions exhibited morphologic a n d metabolic properties o f proximal tubules. This m e t h o d was later extended to rat, 2-4 dog, 5 mouse, 6 a n d h u m a n 7 kidney. This preparation was morphologically d e m o n s t r a t e d to contain m o r e t h a n 90% proximal tubules. 8 An additional Percoll gradient step allows further
~aM. B. Burg and J. Orloff, Am. J. Physiol. 203, 327 (1962). 2N. Nagata and H. Rasmussen, Proc. Natl. Acad. Sci. U.S.A. 65, 368 (1970). 3 W. G. Guder, W. Wiesner, B. Stukowski, and O. Wieland, Hoppe-Seyler's Z. Physiol. Chem. 352, 1319 (1971). + W. G. Guder, Biochim. Biophys. Acta 584, 507 (1979). 5G. Baverel, M. Bonnard, E.d'Armagnac de Castanet, and M. Pellet, Kidney Int. 14, 567 (1978). 6 G. Wirthensohn and W. G. Guder, Pfluegers Arch. 404, 94 (1985). 7 G. Baverel, M. Bonnard, and M. Pellet, FEBS Lett. 101, 282 (1979). s W. Pfaller, W. G. Guder, G. Gstraunthaler, P. Kotanko, J. Jehart, and S. Pfirsehel, Biochim. Biophys. Acta 805, 152 (1984).
[ 18]
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327
purification9 and separation of a small fraction of pure distal convoluted tubule segments.~° This preparation can be applied to all metabolic studies of function which have been found to be preferentially, if not exclusively, localized in the proximal tubule. Renal gluconeogenesis, amino acid, lipid, and ketone body metabolism, as well as oxygen consumption and hormonal regulation and degradation exhibited optimal rates when studied in this model.H In addition this preparation can be used to study intracellular compartmentat i o n $'12 as well as membrane composition of proximal tubules? 3 Medullary Tubules. More recently a similar technique has been applied to prepare tubules from the outer and inner medulla? 4,~5 Outer medullary tubule suspensions consist mainly of thick ascending limb segments, contaminated with other tubule types and cellular debris which can be removed by density gradient centrifugation on Percoll leading to a suspension of 96% thick ascending limbs.14 When studying oxygen consumption, substrate metabolism, and hormone action, they establish properties of the mitochondria-rich cells of the ascending limb of Henle's loop. Inner medullary tubule suspensions consist mainly of collecting tubule segments with some thin limbs and can be used as a model for studying metabolism of the renal inner medulla. ~5
Isolated Cells With the aid of calcium chelators [ethylenediaminetetraacetic acid (EDTA), citrate], tight junctions connecting tubule cells can be weakened to obtain isolated cell suspensions. These were prepared from rabbit renal cortex 16 and outer medulla 17 and further purified by free-flow electrophoresis. ~sThis procedure led to proximal and distal tubule cells of high purity, whose metabolic functions, however, were not so well maintained compared to tubule suspensions. ~9 Further purification of distal cell types can 9 D. W. Scholer and I. S. Edelman, Am. J. Physiol. 237, F350 (1979). ~op. Vinay, A. Gougoux, and G. Lemieux, Am. J. Physiol. 241, F403 (1981). H G. Wirthensohn and W. G. Guder, Physiol. Rev. 66, 469 (1986). ~2W. G. Guder and S. POxsehel, Int. J. Biochem. 12, 63 (1980). 13C. LeGrimellec, M.-C. Giocondi, B. Carriere, S. Carriere, and J. Cardinal, Am. J. Physiol. 242, F246 (1982). t4 M. E. Chambedin, A. LeFurgey, and L. J. Mandel, Am. J. Physiol. 247, F955 0985). ~5G. Wirthensohn, F. X. Beck, and W. G. Guder, PfluegersArch. 4109,411 (1987). ~6p. Poujeol and A. VandewaUe,Am. J. Physiol. 249, F74 (1985). ~7j. Eveloff, W. Haase, and R. Kinne, J. CellBiol. 87, 672 (1980). n A. Vandewalle, B. Ktpfer-Hobelsberger, and H.-G. Heidrich, J. Cell Biol. 92, 505 (1982). ~9H.-G. Heiddch, A. Vandewalle, B. Ktpfer-Hobelsberger, and W. G. Guder, in "Cell Function and Differentiation" (A. Evangelopoulos, ed.). Liss, New York, 1982.
328
KIDNEY
[18]
be obtained by immunoabsorption and lectin absorption techniques2°-22 and can be used as a basis for metabolic studies and cell culture techniques.
Microdissection Procedures At the beginning of this century methods were developed to isolate single nephrons.23 This maceration technique was done by treatment of the tissue with 75% hydrochloric acid. Although proteins were denatured, well-preserved epithelial structures could be obtained. To isolate discrete nephron segments for the study of enzyme activities or metabolic pathways, however, two different techniques have been applied and have become well established in the past 20 years. Microdissection from L yophilized Tissue Sections. This technique, initially developed by Linderstrom-Lang et al.U for metabolic studies in microbial cells, was extended and amended by O. Lowry and associates25 for microdissection of freeze-dried tissue sections. This technique is well suited to the isolation and identification of single tubular structures from freeze-dried renal sections.26 In this procedure fresh kidney tissue is quickly cut into small cones of 2-mm thickness, mounted on a tissue holder, and plunged into liquid nitrogen to maintain structural and biochemical integrity and reduce the development of ice crystals. 27 The tissue sample is transferred to a cryostat and sections of 16-#m thickness are cut with a microtome. This procedure preserves the fuli activity of most enzymes (for exceptions, see Pros and Cons of the Microdissection Procedures, below). Single segments are dissected using an alternate periodic acid-Schiff base-stained section as a guide.26 Single tubular structures obtained from these tissue sections are weighed on a quartz fiber balance calibrated with quinine crystals. 26 Dry weights of about 1 - 5 0 ng can be quantified with this method. 2oM. Le Hir and U. C. Dubach, Histochemistry 74, 521 (1982). 21 W. S. Spielman, W. K. Sonnenberg, M. L. Allen, L. J. Arend, K. Gerozissis, and W. L. Smith, Am. J. Physiol. 251, F348 (1986). 22A. Vandewalle, M. Tauc, F. Cluzeaud, P. Ronco, F. Chatelet, P. Verroust, and P. Poujeol, Am. J. Physiol. 250, F386 (1986). 23K. Peter, "Untersuchungen tiber Bau und Entwicldung der Niere." Gustav Fischer Verla~ Jena, German Democratic Republic, 1909. u K. Linderstrom-Lang, H. Holter and A. S. Ohlson, C.R. Lab. Carlsberg, Ser. Chim. 21,315 (1938). 22O. H. Lowry, J. Histochem. Cytochem. 1, 420 (1953). 26U. Schmidt and U. C. Dubach, Prog. Histochem. Cytochem. 2, 185 (1971). 27U. Schmidt, U. C. Dubach, W. G. Guder, B. Funk, and K. Pads, in "Biochemical Aspects of Kidney Function" (S. Angielsld and U. C. Dubach, eds.), p. 22. Huber, Bern, Switzerland, 1975.
[ 18]
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[18]
The amount of tissue needed depends on the sensitivity of the assay procedure and the activity/concentration of the measured enzyme/substrate in the respective segment. Microdissection from Fresh Tissue. This method, first described by Burg and Odoff in 1962,~" is based on the free-hand preparation of single nephron segments from fresh renal tissue. The procedure is, however, limited to kidneys of animals with easily accessible connective tissue, as is found in rabbit kidney. To separate different medullary and distal nephron structures in other animals, collagenase pretreatment of the kidney is needed. This disintegration of renal connective tissue by collagenase was extensively examined by Morel and co-workers2s and has been applied by many different groups29-33 to overcome the problem of intranephron heterogeneity. For the preparation of single-tubule samples, kidneys are perfused with isotonic saline supplemented with glucose, bovine serum albumin, and collagenase. 28,~,35 After ligation of the renal vein and the ureter the same solution is injected under pressure until rupture of the renal capsule. The kidney is sliced along the corticomedullary axis and small pyramids are incubated at 35-37* for 20-40 min (depending on the animal) in the same collagenase buffer bubbled with a stream of air. The small tissue pyramids are washed with collagenase-free buffer and microdissection is carried out with fine needles under a stereomicroscope. Each dissected nephron segment is photographed for confirmation of identity and measurement of tubular length. Figure 2 shows some representative photographs of tubular segments of rabbit nephron.
Advantages and Limitations of the Various Isolation Procedures Tubule Suspensions. This preparation has the great advantage that many identical samples of relatively well-defined segments can be studied. Routine methods may be applied for metabolite, oxygen, and enzyme activity measurements. Compared to isolated cells and tubule-derived cell 28M. Imbert, D. Chabard~s, M. Monttgut, A. Clique, and F. Morel, Pfluegers Arch. 354, 213 (1975). 29A. Doueet, A. I. Katz, and F. Morel, Am. J. Physiol. 237, FI05 (1979). 30H. Endou, H. Nonoguehi, J. Nakada, Y. Takehara, and H. Yamada, in "Kidney Metabolism and Function" (R. Dzurik, B. Lichardus, and W. G. Guder, ¢kls.) p. 26. Nijhoff, Boston, Massachusetts, 1985. 3~R. M. Edwards, B. A. Jackson, and T. P. Dousa, Am. J. Physiol. 238, F269 (1980). 32N. Farman, A. VandewaUe, and J.-P. Bonvalet, Am. J. Physiol. 244, F325 (1983). 33G. Wirthensohn, A. VandewaUe, and W. G. Guder, Kidney Int. 21, 877 (1982). F. Morel, Am. J. Physiol. 240, F159 (1981). 35A. Vandewalle, G. Wirthensohn, H.-G. Heidrieh, and W. G. Guder, Am. J. Physiol. 240, F492 (1981).
[ 18]
METABOLISM OF ISOLATED TUBULE SEGMENTS
331
cultures a high degree of viability and differentiation is maintained. On the other hand, several limitations have to be considered when using these preparations. Besides effects of collagenase, preparations are always contaminated with minor fractions of other cell types. This can be especially disturbing if isotopes are applied to study metabolic pathways. Likewise minor contaminations can lead to major errors if the studied enzyme is very low in the main cell type.36 In addition, metabolite levels may change during preparation and therefore must be compared with the in vivo levels. Pros and Cons of the Microdissection Procedures. A great advantage of the "Lowry" technique is the low amount of tissue needed for preparation (< 10 mg fresh weight). Moreover, the rapid freezing of the tissue in liquid nitrogen preserves the in vivo conditions. This is of special importance for the study of human tissue, which is available only as biopsy or after nephrectomy. The technique allows storage of tissue in a deep-frozen state over several months to years before dissection is performed. In the hands of a well-trained person this microdissection procedure provides a high degree of flexibility and accuracy. Limitations, on the other hand, arise mainly from the fact that this isolation technique does not provide full integrity of tubular cells. For this reason metabolic pathways and hormone actions cannot be studied. Moreover several enzymes, such as pyruvate carboxylase37 and adenylate cyclase (W. G. Guder, unpublished observations), lose their activity on lyophilization and/or freezing. In contrast to fresh tissue dissection the former method does not allow the dissection of different segments in one tubule, offering the possibility of studying intranephron heterogeneity. Furthermore, only fresh tissue dissection is suitable for studying the sites of hormone actions~,2s,34,38and metabolic pathways such as tdacylglyceroP9 and glucose4° synthesis as well as CO2,41'42 NH 3,43,44 and lactate45 formation.
W. G. Guder and A. Rupprecht, Eur. J. Biochem. 52, 283 (1975). 37 H. B. Butch, R. G. Narins, C. Chu, S. Fagioli, S. Choi, W. McCarthy, and O. H. Lowry, Am. J. Physiol. 235, F246 (1978). 38F. Morel and A. Doucet, Physiol. Rev. 66, 377 (1986). 39W. G. Guder and G. Wirthensohn, in "Biochemistry of Kidney Functions" (F. Morel, ed.), p. 95. Elsevier, Amsterdam, 1982. 40A. Maleque, H. Endou, C. Koseki, and F. Sakai, FEBS Lett. 166, 154 (1980). 41 F. LeBouffant, A. Hus-Citharel, and F. Morel, PfluegersArch. 401, 346 (1984). 42F. LeBouffant, A. Hus-Citharel, and F. Morel, in "Biochemistry of Kidney Functions" (F. Morel, ed.), p. 363. Elsevier, Amsterdam, 1982. 43 H. Nonoguchi, S. Uchida, T. Shiigai, and H. Endou, PfluegersArch. 403, 229 (1985). 44 D. W. Good and M. B. Burg, J. Clin. Invest. 73, 602 (1984). 45S. Bagnasco, D. Good, R. Balaban, and M. Bur~ Am. J. Physiol. 248, F522 (1985).
332
KIDNEY
[ 18]
The disadvantage of fresh tissue dissection lies in the use of collagenase, which may alter tubular membrane permeabilities and affect enzyme activities. 33,35,46In addition the material is of limited use in the measurement of metabolites since the preparation and incubation procedure may change their concentrations. If regulation in vitro is, however, to be studied at the single-nephron level, freshly dissected tubules are adequate. Microanalytical Procedures To study metabolite concentrations, enzyme activities, hormone actions, or metabolic pathways in single-nephron segments obtained by the microdissection procedures described, sensitive microassays are needed which allow the detection of specific products/metabolites in the picomolar range. This can be achieved by either of the following techniques, which have been successfully applied to renal tissue. Considerable increase in sensitivity can be obtained by decreasing assay volumes to the nanoliter range. This tiny volume is pipetted into small holes (4 × 3 mm) of a Teflon rack sealed with a drop of paraffin oil (oil well) to prevent evaporation.47 Reactions in a few microliters can be performed between two glass slides with opposed depressions, an arrangement allowing radiochemical41,42and cycling3° procedures to be performed. Table I summarizes enzymes, substrates, and pathways which have been successfully analyzed with one of the following techniques.
Enzymatic Cycling Enzymatic cycling analyses were performed with methods based on specific enyzmatic reactions which result in oxidation or reduction of pyridine nucleotides.47 Amplification of signals is obtained by use of enzymatic cycling of nucleotides.48 The following examples illustrate the principles of enzymatic cycling as it is used to study enzyme activities or renal metabolites. Citrate Synthase 49. The tissue to be studied is added to a reagent that carries out the following reaction: Oxaloacetate + acetyl - CoA ---, citrate + CoA 4~ G. Wirthensohn, A. VandewaUe, and W. G. Guder, Biochem. J. 198, 534 (1981). 47 O. H. Lowry and J. V. Passonneau, "A Flexible System of Enzymatic Analysis," Academic Press, New York, 1972. 4s O. H. Lowry, Mol. Cell. Biochem. 32, 135 (1980). 49 H. B. Butch, in "Biochemistry of Kidney FUnctions" (F. Morel, ed.), p. 297. Elsevier, Amsterdam, 1982.
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336
KIDNEY
[18]
After oxaloacetate is destroyed by addition of NaOH and heating, a solution is added containing reagents which catalyze the following reaction: Citrate
, acetate + oxaloacetate NADH ~
_malate NAD +
Excess NADH is destroyed by hydrochloric acid and the following cycle is carded out for 60 min: Malate. NAD + _ ethanol
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The cycling enzymes are malate dehydrogenase and alcohol dehydrogenase. The cycle is stopped and malate measured in the indicator reaction with the malate dehydrogenase and aspartate aminotransferase reagent: glutamate~_
