Correlation of Structure and Active Transport in the Teleost Nephron BENJAMIN F. TRUMP AND RAYMOND T . JONES Comparative and Environmental Pathobiology Program of t h e Department of Pathology, Uniuersity of Maryland School of Medicine, Baltimore, Maryland 21 201
ABSTRACT In the present study we have extended our investigations concerning the correlation between ultrastructure and active transport in the isolated flounder nephron. The composition of the fish nephron is defined in ultrastructural terms and its behavior when incubated in vitro under short term and long term culture conditions is described. Using the in vitro system originally described by Forster, a variety of inhibitors and conditions which modify cell structure and function were tested. Ultrastructure was correlated with chlorphenol red dye transport. In general, conditions altering active transport also markedly altered cellular ultrastructure. The principal alterations consisted of membrane changes involving various organelles -most importantly the plasma membrane and the mitochondria. Conditions associated with irreversible cell injury could be rapidly produced by interference either with mitochondrial ATP synthesis or with the integrity of the plasma membrane. Both of these rapidly lead to irreversible events which are preceded by reversible structural changes. Organelle changes progress in a rather well-defined sequence of reversible and irreversible stages which are defined. One difference between the two types of interactions is the presence of intramitochondrial calcification which does not occur with direct modification of the mitochondrial electron transport system. The concept of utilizing long term explant organ cultures of fish nephrons for environmental studies is introduced.
The purpose of this paper is to present a synthesis of studies performed in our laboratory on the relationship between ultrastructure and active transport i n the fish nephron. We will attempt to correlate the effects of a variety of extrinsically induced interactions on structure and active transport in the teleost nephron-briefly touching on some that have been reported previously yet expanding this to conditions not heretofore described and finally attempting a synthesis of this information into an integrated picture of the subject. The history of active transport studies in the fish nephron spans several decades and has enriched the general biology of transport in general and of fish and mammalian nephron transport in particular. Studies on the system reported here were first introduced by Forster ('48) i n his paper on the use of isolated nephrons and thin kidney slices for studying active transport. In a brilliant series of studies, Forster and his colleages ('53, '54, '56, '57, '58, J. EXP. ZOOL.,199: 365-382
'61, '67) systematically explored many of the characteristics of the transport systems and determined many of their metabolic requirements. These studies, supplemented by those of Puck et al. ('52) and Wasserman et al. ('53) developed the concept of a two-stage system, one involving entry from the suspending medium into the cells and the second, from the cells into the tubule lumen. Later Kinter ('66) using microspectrophotometry and radioactively labelled chlorphenol. red began to develop more quantitative estimates and kinetic analyses of transport in these systems and Burg and Weller ('69) using isolated perfused tubules further characterized the process. In our laboratory we began i n the late fifties to develop a n ultrastructural understanding of the nephron and to attempt correlation between changes in ultrastructure and the active transport process (see review by Trump and Bulger, '71). As a spin-off of that work we began to define the regional characteristics of the fish 365
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BENJAMIN F. TRUMP AND RAYMOND T. JONES
nephron and several years ago, together with Hickman (Hickman and Trump, '69) attempted to integrate all available morphologic and functional information on the kidneys of fish of all types. All of these fish studies have had many implications for biomedical science. They have been of obvious direct importance to the understanding of fish physiology and have contributed substantially to the elucidation of renal structure and function in the human. The studies have also contributed substantially to the general cell biology of active transport and moreover have made essential contributions to the development of modern cellular pathology, since cellular reaction to injury as it is now known, was in large part developed by the study of these isolated fish nephrons with which controlled experimentation in vitro could be performed (Trump and Ginn, '69). Most of the conclusions derived from earlier studies have stood us in good stead in subsequent studies on mammalian disease at the ultrastructural level including human disease. MATERIALS AND METHODS
Specimens of three species of flatfish were utilized in these studies. All were obtained by either dragging or gill netting and maintained in sea water aquaria or artificial sea water aquaria at approximately 14" C. Species utilized were Porophrys uetulus, the English sole from Puget Sound near Seattle, Washington; Paralichthys lethostigma, the Southern flounder from the estuaries near Beaufort, North Carolina; and Trinectes maculatus, the hogchoker, from the Potomac River near Morgantown, Maryland and the Patuxent River near Benedict, Maryland. In all cases the preparations were obtained by sacrificing the fish quickly, removing the kidney and immersing it in cold (0-4" C) Forster's buffer with the following composition in mMoles per liter; NaCl, 135; KC1, 2.5; CaC12, 1.5; MgC12, 1.0; NaH2P04, 0.5; NaHC03, 10. Small fragments measuring a few mm in diameter were quickly cut with scissors and these were, in turn, teased with dissecting needles into separated clumps of tubules. These were maintained in Forster's buffer at 0-4" C until used. Two types of incubation were carried
out. For short term incubation the tubules were incubated in suspensions in Petri dishes or small centrifuge tubes and gassed by bubbling oxygen through the medium via a 21 gauge hypodermic needle. For long term incubations the tubules were placed in 60 mm plastic Petri dishes containing CMRL 1066 with 1 pg insulin per ml, 0.1 pg hydrocortisone hemisuccinate per ml, 2 mM L-glutamine, 300 U penicillin G per ml, 300 pg streptomycin per ml, and 2.5 pg Amphotericin B/ml, 5 % heat-inactivated fetal calf serum. The dishes were maintained in a controlled atmosphere chamber and gassed with a mixture of 45% 02,50% N2 and 5 % COz. The chamber was rocked at 10 times per minute and maintained at 37" C. Transport was studied by incubation in media containing 1 X 10-4 molar chlorphenol red and by periodic observation of the tubule clumps by light microscopy. The transport was noted as intraluminal accumulation which was scored as follows: 0 , no accumulation; 1 coloration just detectable; 2 , intermediate coloration; and 3 , intense coloration. Tissues were fixed for light and electron microscopy in either 1% osmium tetroxide buffered with s-collidine, 4 % glutaraldehyde, or a mixture of 4 % formaldehyde and 1 % glutaraldehyde in 0.2 molar phosphate buffer. The aldehyde fixed tissues were washed and post-fixed in osmium tetroxide. All tissues were dehydrated in ethanol and embedded in Epon. Thin sections were cut and double-stained with uranyl magnesium acetate and lead citrate. Compounds were added directly to the incubation media in the final concentrations noted in the results and in the table. In case of lipid soluble inhibitors these were dissolved in a small amount of ethanol. Controls were run containing similar amounts ofethanol.
+
+
+,
Fig. 1 Electron micrograph of hogchoker kidney tubule from the second brush border segment incubated in oxygenated Forster's buffer for 1 hour. Note the excellent preservation of cellular structure. The lumen (L) is at the top; the basement membrane (BM) is a t the lower left. Note the appearance of nucleus, mitochondria (M), basilar infoldings of cell membrane (free arrow), apical junctional complexes (JC) and intracellular membranes including endoplasmic reticulum. A small amount of cell debris is in the lumen at the top. Incubated flounder nephrons maintain this type of ultrastructure for many hours. x 5,000.
STRUCTURE A N D FUNCTION OF FISH NEPHRONS
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BENJAMIN F. TRUMP AND RAYMOND T. JONES RESULTS
The marine teleost nephron The nephron of the typical marine teleost appears to consist of the following segments beginning after the renal corpuscle (Bulger and Trump, '68): a neck segment, a first proximal segment, a second proximal segment which sometimes has a terminal third segment, a collecting tubule, and a collecting duct. A typical distal segment resembling that in mammals is not seen in the marine teleosts thus far studied in detail; nor do they possess a ciliated intermediate segment between the second or third proximal segment and the distal or collecting tubule. The following is a description of the typical features of the several segments. A renal corpuscle with juxtaglomerular cells is typically present (Bulger and Trump, '69a). The neck segment is made up of ciliated cuboidal cells that contain many mucous granules. The next segment of the nephron is the first proximal segment (PSI). This segment is composed of columnar cells with a well-developed cytocavity network. Numerous large lysosomes as well as large mitochondria are present. Closely packed microvilli along with apical tubules at their base are further identifying features of this segment. The cells of proximal segment I1 are similar to those of PSI, however, they are somewhat taller and the microvilli are sparser. Mitochondria are scattered throughout the cell and the basilar membranes form a well-developedlabyrinth. The third proximal segment, when present, consists of similar, although shorter, cells with an abundant endoplasmic reticulum (ER). The collecting tubule cells also have apical mucous granules, but only a few microvilli and an occasional cilium. The collecting tubules converge into a collecting duct system whose cells are surrounded by smooth muscle cells (Hickman and Trump, '69; Trump and Bulger, '71). Control tubules in uitro; Short-term When control tubules are incubated in Forster's buffer and gassed with oxygen, active transport proceeds briskly with marked intraluminal accumulation as previously noted by Forster and good preservation of ultrastructure (Trump and Bulger, '67). Transport appears to occur in all proximal segments. It seems unlikely that the neck transports but it is difficult to
state this with certainty. It does not appear that transport occurs in the collecting ducts. Morphologically, the tubules, which are largely collapsed at the time of removal from the fish with blebs of cytoplasm into the lumen, inflate during the incubation and maintain good preservations for more than 8 hours (fig. 1). Using these incubation conditions there is good preservation of mitochondria, cell membranes, nucleus, endoplasmic reticulum, Golgi apparatus and lysosomes throughout the period. The only significant ultrastructural changes that occur are the formation of numbers of lysosomes containing degenerative organelles such as mitochondria. Many of these appear to result from autophagocytosis, as previously described, (Trump and Bulger, '67) involving sequestration of organelles by wrapping with ER cisternae forming autophagic vacuoles (AV). Some of the larger AV may also have contributions from phagocytosis of debris resulting from damaged tubules passing down the lumen and phagocytosis by the epithelium. It is apparent that this increased organelle turnover and accumulation of AV is not associated with impairment of active transport. It is also not clear whether the accumulation of AV results from an increased rate of formation in incubated tubules or from a decreased rate of turnover andlor extrusion of contents from these bodies. Also, systems for rapid transport of colloidal particles from lumen to both exist (Bulger and Trump, '6%).
Control tubules in uitro; Long term When the tubules are cultured under the organ culture conditions previously characterized for mammalian bronchi and pancreatic duct (Barrett et al., '76) good viability can be maintained for as long as Fig. 2 Electron micrograph of hogchoker kidney culture maintained i n CMRL 1066 medium for 15 days. The lumen (L) is at the top, the basement membrane area toward the lower right. The lack of a well-defined basement membrane in this area may indicate that this is one of the outgrowths of tubular structures that occur at the margins of these explant cultures. Note the appearance of the cell which is compatible with continued viability. However, certain changes can be clearly identified such a s the marked shape change i n the mitochondria which are frequently bowlor doughnut-shaped, presence of annulate lamellae (arrow), and numerous residual bodies (RB) or secondary lysosomes presumably derived from extensive autophagocytosis. Note preservation of junctional complexes along the lumen. X 5,000.
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TABLE 1
w e c t s of various c o m p o u n d s and altered conditions on active transport Condition
Conc.
LET to Zero
Condition
Conc.
LET to Zero
+
~
Mitochondrial znhibztors CN 3 x 10-3 Antimycin 2x
Azide Oligomycin BAL
Tvrocidine 2,4 DNP
10-3 10-4 10-4 10-5 10-2 10-3 10-4 10-5 10-2 10-3 10-4 10-5 10-3 10-4 10-3 10-4 10-5
Continuous 15 min 30 min 1 hr 2 hr 4 hr
N2
Amytal
10-3 10-4 10-5
Glycolytic inhibitors IAA
KF
10-3 10-4 10-5 10-3 10-4 10-5
Membrane active c o m p o u n d s Ouabain 10-2 10-3 10-4 10-5 10-3 10-4 10-5 12.5 ygtml 1.25 yglml
Gramicidin Amphotericin
.1mglml
Filipin
.01 mg/ml 10 mg%
Vitamin A
20 mg% 2 gm%
Saponin Digitonin
10-4 10-5
CPC
10-3 10-4 5 x 10-4
Chlorcquine
5 x10-5
5 x 10-6
Phospholipase
.2 mglml .02 mglml
Modification of m e d i u m Mg-free Ca free EDTA K-free PH-3 pH-9.3
24 hr 30 min-0 30 min-0 30 min-0 15 min-0 4 hr-0 2 hr-0 No effect 2 hr-k 6 hr-? 30 min-0 30 min-0 Minimal effect No effect 30 min-0 Minimal 15 min-0 15 min-0 Minimal effect 2 hr-1 No effect No effect 1hr-2+ 2 hr-2 4 hr-0 0 0-s1igh t Very slight
+
+
15 min-0
2 hr-0 No effect No effect No effect 1hr-0 30 min-0
4 hr-0 4 hr-0 4 hr-2 No effect No effect 4 hr-0 4 hr-O-l+ 2 hr-0 2 hr-0 No Inhib. 6 hr-+ 15 min-0 4 hr-0 6 hr-0 5 min-0 5 min-0 4 hr-0 No effect No effect 15 min-0 1 hr-0
+
+
20 c 5 hr
10 - 2 1.8 X 10-2 3.5 X 10-2 6.9 X 10-2 1.4 X 10-1
+
Hypotonia .75 Forster's .5 Forster's
Normal Normal 30 min-0 5 min-0
'25 Forster's Dist. HzO
Heauy m e t a l s Arsenate Arsenite PCMB
10-2 10-3 10-4 10 - 2 10-3 10-4 10-2 10-3 10-4 10-5
PCMBS
10-3 10-4 10-5
Hgaz
10-3
1hr-0
CHTHgCI
CdS04
10-4 10-5 10-6 10-3 10-4 10-5 10-6 10-3
10-4
Pb citrate ZnClZ
10-5 10-6 10-7 10-4
_7 _0 - 5
10-6 10-3 10-4 10-5
Inhibitors of macromolecular synthesis Puromycin 2 x 10-4 Pactamycin 10-4 10-5 10-6
Actinomycin
50 yg/ml 5 yglm1 10-4
2 hr-0 4 hr-0 4 hr-0 15 min-0 2 hr-+ Minimal 15 min-0 15 min-0 2 hr-0 4 hr-+ 15 min-0 2 hr-0 4 hr-slight effec 15 min-0 15 min-0 4 hr-1 6 hr-2+ 15 min-0 15 m i n d 6 hr-slight effec 6 hr-no effect 15 min-0 15 min+J 15 min-0 1hr-0 4 hr-1 Minimal No effect No effect 15 min-0 Minimal Minimal
+
+
6 hr-No effect No effect No effect No effect 5 hr-+
30 min-+ Cells 3 1 hr-0 No cell or lumen 15 min-0
Chloramphenicol 20 yglml 2 m/ml
No effect No effect No effect 4 hr-+ No effect 4 hr-+ No change
p-fluorophenylalanine
5 hr-0
1 hr-0
Carcinogens fluoro-biphenylacetamide
+
30 min-0
PH-10.5
48 h r 3 days 4 days KCl
Cells-1 4 hr-0 1 hr-0 1hr-0 1 hr-0 1 hr-2 1 hr-+ 2 hr-0 1hr-0-+ 1hr-0
No effect
Streptovitacin
3 X 10-6 Cycloheximide
Ethionine
10-4 10-5
400 yglml 40 yglml 10-2 10-3
10-3 M
5 hr-+ 1 hr-0 1hr-0
Slight inhib. by 1h r
LET, least effective time; BAL, antilewisite; IAA, Iodoacetate acid; KF, Potassium flvoride; CPC, Cetyl... - British . ~
STRUCTURE AND FUNCTION OF FISH NEPHRONS
371
Fig. 3 Basilar portion of tubular epithelial cells of Southern flounder after 4 hours incubation with 1 0 - 4 antimycin. This treatment is associated with complete inhibition of electron transport and chlorophenol red dye transport. The cells undergo early swelling and later become irreversibly altered. Such a stage is shown here. Note the marked swelling of mitochondria (M) with tubular conversions of cristae, lamellar stacks of basilar infoldings (free arrow) and a nucleus (N) showing karyolysis. The endoplasmic reticulum (ER) is dilated. X 5,000. Fig. 4 Isolated Southern flounder kidney tubule incubated 30 minutes with potassium cyanide in the presence of added ATP. ?tyo types of cell change are shown. In the first, shown on the left, the mitochondria are condensed with expansion of the outer compartment, while i n the cell on the right mitochondria show high amplitude swelling. Both of these changes are reversible and the condensed conformation appears to occur earlier i n time as indicated i n the cell stages mentioned in the text. X 15,000.
two weeks, although during this time the tubules maintained an altered form and the ultrastructural findings were associated with good viability (fig. 2). At the edges of the cultured fragments apparent division and growth of cells occurs regenerating a layer of epithelium around the periphery of the clump which in areas begins to form intercellular canaliculi with cell junctions that were suggestive of attempted lumen formation. Cells in these prolonged cultures retained a polarity with junctional complexes toward the luminal surface but alterations occurred within the cytoplasm. Mitochondria changed from their normally elongate configuration to rounded doughnut-shaped profiles often associated with the close apposition of lipid droplets. Close packing of cristae and lamellar formations between cristae were also observed.
Inhibition of Oxidative metabolism
In the table is shown the effects of a variety of inhibitors of mitochondria1 function including inhibitors of the electron transport system, inhibitors of oxidative phosphorylation, and uncouplers of oxidative phosphorylation. M e n these compounds were tested, at the concentrations indicated, on isolated transporting tubules, inhibition of active transport was noted fairly rapidly in all cases. The molar concentrations required for maximum inhibition varied as indicated. In general, the inhibition was initially reversible in the sense that when the tubules were removed from the inhibitor-containing medium and replaced into normal medium with oxygenation, transport was again reinstituted. This phase lasted from 30 minutes to 2
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BENJAMIN F. TRUMP AND RAYMOND T. JONES
STRUCTURE AND FUNCTION OF FISH NEPHRONS
373
Fig. 6 Isolated English sole tubule incubated 6 hours i n medium containing 1 0 - 4 M 2,4-dinitrophenol. This is a first brush border segment showing numerous lysosomes characteristic of this region, but showing irregularities and slight condensation of mitochondria typical of early mitochondrial changes following uncoupling or inhibition of respiration. X 5,000.
hours. The most useful condition for studying this type of reversibility was bubbling with purified nitrogen since in that case one is not concerned with removal of residual toxic compounds even in the replacement medium. During the course of incubation, light microscopy using phase or Normarski optics revealed that the cells consistently underwent softening of contours, swelling, and obliteration of lumens. With careful studies at the longer time intervals it could be seen that all mitochondria within the Fig. 5 Tubule from the first brush border segment of a n English sole incubated for 1 hour in medium gassed with purified nitrogen. This was associated with inhibition of dye transport which was reversible at this stage. Note the mild clumping of chromatin (Ch), and tiny flocculent densities (free arrow) i n the mitochondria. Both of these are known to be reversible in this system and subsequently have been found to be reversible alterations in mammalian systems as well. X 6,500.
epithelial cells had undergone swelling to form a population of spherical profiles in the cvtotdasm. Election microscopic studies of the progression of change following such inhibitors revealed a fairly reproducible sequence of stages through which the cells pass between the beginning or 0-time state and the final irreversible or necrotic state (fig. 3 ) (Trump and Bulger, '68a,b). Furthermore, by comparing these stages with the point of loss of reversibility the reversible ones (figs. 4 and 5) could be distinguished from those which were irreversible. For purposes of the discussion we have arbitrarily numbered these stages from 1 , the normal cell, to 7, the advanced necrotic cell (Trump and Ginn, '68; Trump et al., '76). In stage l a the nuclear chromatin is clumped and the mitochondrial granules disappear along with a reduction in cell glycogen. Cells of stage 2 of cell injury
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STRUCTURE AND FUNCTION OF FISH NEPHRONS
have dilated ER, distorted plasma membranes and slightly swollen lysosomes. Stage 3 is characterized by condensed mitochondria and expanded cell sap (fig. 4) while in stage 4 some of the mitochondria are swollen while others are condensed. The mitochondria are all swollen in stage 4a and there is some loss of the polysome arrangement. In stage 4b, tiny flocculent densities are present in the mitochondria. Flocculent mitochondrial densities are the hallmark of stage 5 . Stage 5a is similar to stage 5 ; however, mitochondrial calcifications are present. Lysosomes have lost their integrity in stage 6 while in 6a there are mitochondrial calcifications. As mentioned above, stage 7 represents the completely necrotic cell in which large myelin forms have formed along with karyolysis and fragmented cell membranes. Stage 7a has mitochondrial calcifications. Stages 1 through 4 b of cell injury appear to be reversible while stages 5 through 7a appear to be irreversible (Trump and Arstila, '75). The changes indicated above in stages 2 through 7a are cumulative. It is noteworthy that in none of these conditions did mitochondrial accumulations of calcium phosphate occur (fig. 6). This is in contradistinction to some of the other conditions noted below. The difference is apparently the lack of active mitochondrial calcium uptake in the presence of a potent mitochondrial inhibitor or uncoupler.
375
tively little effect was noted. This may be due to the fact that ATP can be formed in the presence of fluoride and also because other substrates such as fatty acids may be utilized in addition to those arising from the glycolytic sequence. Since prominent glycogen deposits are not present in flounder nephrons, the latter seems especially likely. Iodoacetate had very severe effects similar to those of inhibition of oxidative metabolism above; however, it may well be that the effects are due to changes other than in the glycolytic sequence such as the effect of iodoacetate on membrane sulfhydryl groups.
Cell membrane interactions Over a fairly wide series of concentrations ouabain treatment was associated with inhibition of dye transport and cell swelling with direct reproduction of the phases described under oxidative inhibition above. A very important difference was that in this case mitochondrial calcification did occur (fig. 7). It was further noted that respiration persisted in the presence of ouabain. The cell swelling was often extremely marked.
PCMB and PCMBS The organic mercurials also resulted in marked inhibition of transport and also in rapid cell swelling and necrosis (over a fairly wide range of concentrations) (Sahaphong and Trump, '71). The fact that changes were similar with PCMBS (paraGlycolytic inhibitors chloromercuribenzenesulfonate), a relativeMixed results were obtained with these ly non-penetrating mercurial and PCMB compounds. With fluoride at fairly high (parachloromercuribenzoate), suggested concentrations as shown in the table, rela- that the most important interaction was involved at the surface membranes. We also Fig. 7 Southern flounder kidney tubule from the second brush border region incubated in the noted a potential reversibility of the transpresence of 1 0 - 3 M ouabain for 2 hours. Note that port defect with the PCMB and PCMBS as most mitochondria are swollen while one is con- observed with the oxidative inhibitors (durdensed (arrow). The swollen mitochondria contain ing the first few minutes). Also, as with densities (D) adjacent to the inner membrane and membranes of the cristae which can be shown to the ouabain, mitochondrial calcification represent intramitochondrial calcium phosphate was seen except with the highest concenaccumulations. This cell is probably irreversibly trations. This again is presumed to indialtered. Note dilatation of ER and enlarged, swol- cate lack of penetration of the mercurial len cell sap. X 10,000. into the cells since isolated mitochondria Fig. 8 Southern flounder tubule incubated in the presence of amphotericin (1.3 mglml) for 6 incubated with PCMB showed marked inhours. This treatment is associated with inhibition hibition of calcium uptake (Trump et al., of dye transport and swelling of some mitochondria '75a). Both HgClz and CH3-HgCl2had simiand condensation of others. Note the large intracytoplasmic vacuoles and obliteration of basilar lar, even more rapid effects on both transport and ultrastructure. membrane conformations. X 5,000.
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BENJAMIN F. TRUMP AND RAYMOND T. JONES
STRUCTURE AND FUNCTION OF FISH NEPHRONS
Other agents
Treatment of isolated tubules with ionophores such as amphotericin and filipin also resulted in rapid cytolysis (fig. 8). This was preceded by condensation of mitochondria similar to that which was observed previously with amphotericin treatment of toad bladder. Direct treatment with various surface active agents including saponin, digitonin, cetylpyridinium chloride, and phospholipase all resulted in marked inhibition. With surface saponin (fig. 9), direct dissolution of basilar cell membranes could be noted. Modification of the m e d i u m Elimination of magnesium from the medium was associated with marked retardation of transport with inhibition complete by 1 hour. Electron micrographs showed first condensation and then later swelling of mitochondria. Ultimately, the pattern was similar to that observed with the oxidative inhibitors. Incubation in calciumfree medium or calcium-free medium in the presence of EDTA produced marked alterations of both structure and transport (Bulger and Trump, '69b). With time cells began to separate and soon even by light microscopy the pressure of a cover-slip on the preparation caused extrusion of cells from the tubules leaving the basement membrane cylinders intact. By electron microscopic study complete dissociation of all types of junctions including tight, intermediate, desmosomes and gap junctions was noted. Large channels then existed between the lumen and the basement membrane. In addition, there was marked disorganization of intracellular organelles with complete disorientation of cell apices. Fig. 9 Tubule from Southern flounder incubated in the presence of saponin for 4 hours. Note dissolution of basilar infoldings (free arrow) and marked swelling of mitochondria (M) and other membrane systems such a s the ER. Organelles are difficult to recognize. There is virtually immediate cessation of active transport. Basement membrane, BM. x 8,000. Fig. 10 Isolated tubule from second brush border segment of English sole incubated i n medium devoid of potassium for 30 minutes. This is associated with neither intracellular nor intraluminal accumulation. Note cell sap swelling and marked change of basilar infoldings, some of which (free arrows) appear to have pulled away completely from the basilar cell membrane. x 10,000.
377
The cells tended to undergo enspherulation. Half-desmosomes could be observed along lateral cell margins. No intraluminal dye accumulations could be noted under these conditions although intracellular accumulation still occurred at least during the early stages. Incubation of tubules in potassium-free medium results in neither intraluminal nor intracellular accumulation. There was, however, some cell swelling with marked distortion of basilar cell membranes leaving large basilar compartments with only a relatively simple plasma membrane (fig. 10) (Bulger and Trump, '69b). Replacement of sodium with potassium chloride had the following effects. At 10 and 18 mM potassium there was only slight inhibition of dye transport. At 35, 69 and 140 mM potassium there was marked inhibition complete by 1 or 2 hours. Furthermore, at these latter three concentrations there was marked cell swelling. This was preceded by a phase in which mitochondria were condensed but ER and cell sap were dilated. The condition reproduced the stages described above (Trump and Ginn, '68). Modification of Forster's buffer to produce hypotonic stress and study of volume regulation had the following results. At three-fourths strength Forster's buffer adaptation appeared to occur with both normal morphology and normal dye transport. At one-half strength Forster's buffer, transport was almost normal and many normal tubules occurred for a time. However, at either one-fourth strength Forster's buffer or in straight distilled water no dye transport and marked swelling and cytolysis occurred (fig. 11). At the ultrastructural level the cells rapidly progressed to marked high amplitude mitochondrial swelling. Reduction to pH 3 led to rapid inhibition of transport within the first few minutes. Among the striking ultrastructural effects noted was marked clumping of nuclear chromatin. Elevation of pH to 9.3 or 10.5 also led to inhibition of transport. The main difference at the elevated pH was the pronounced cell swelling that occurred very early and rapid progression to stage 7. Incubation of tubules at 0-4" C followed by reversibly testing led to the observation that cells could tolerate 5 hours of
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STRUCTURE AND FUNCTION OF FISH NEPHRONS
cold incubation with no effect on structure and function. By 24 hours there was retention of transport however with incubations of 2 to 4 hours, transport inhibition was seen. At 3-7 days of cold incubation no transport was seen by 1 hour. Electron micrographs of cells incubated at reduced temperature showed a series of stages comparable to those observed with oxidative inhibition though the rate of change was much slower (fig. 12). Heavy metals A number of heavy metals including arsenate, mercury, cadmium, lead, and zinc have been tested. All of these at some concentration exhibited complete inhibition of dye transport. Mercury and cadmium were among the most toxic of these heavy metals and led rapidly to irreversible cell changes. We are presently comparing the effects of heavy metals on the entire fish as well as on the in vitro tubules. Inhibitors of macromolecular synthesis A variety of inhibitors of macromolecular synthesis have been tested as shown in the table. Although some of these at very high concentrations had slight effects on transport, most had no effect. At the ultrastructural level most changes were compatible with cell survival though some, such as actinomycin, altered polysome and nucleolar morphology (Shelburne et al., '73). Effects of carcinogenic agents So far we have just begun studies of carcinogens in systems maintained for relatively long periods of time in vitro. Our initial studies are with the organic carcinogen fluorobiphenyl acetamide which is known to consistently produce renal adenoFig. 11 Isolated Southern flounder kidney tubule incubated in one-fourth strength Forster's buffer for 30 minutes. Under these conditions cell swelling occurs rapidly with obliteration of basilar processes, enlargement of cell sap, swelling of most mitochondria and dilatation of ER. Some basilar infoldings persist. Volume regulation cannot occur i n this osmolality. Cell swelling and death occur rapidly. X 6,000. Fig. 12 Southern flounder tubule incubated i n Forster's buffer at 0-4" C for 1 week. Under these conditions ability for recovery is lost after approximately 1-2 days. By 1 week irreversible stage 5 changes, including swollen cell sap, swollen mitochondria, flocculent densities and fragmented ER can be seen. X 5,000.
3 79
carcinomas in the rat following long term feeding. This compound, at a concentration of 10-3 M, was associated with inhibition of dye transport although this was never complete. Many tubules showed slight intraluminal accumulation. Ultrastructural studies of the effects of this agent on the tubular epithelium are preliminary but so far indicate a very early effect on tubular cell mitochondria which undergo slight condensation with shape irregularities, formation of ring and doughnut forms, and little other subcellular change. Studies are currently underway testing the longer term effects. DISCUSSION
The unity of the isolated flounder tubule system for the study of cell pathology is shown by the present results as well as those previously published. It is especially suitable for studying the correlation between ultrastructure and active transport as well as for examining a wide variety of cellular reactions to injury. The results of experiments performed in our laboratory over the past 20 years using this system have clearly been of value in determining the relationship between organelle change and cell function and have laid the foundation for further studies on the relationship between ultrastructure and function in injured cells in a variety of higher animal systems including man (Trump and Arstila, '75; Trump et al., '76). Using this system, originally described by Forster ('48), the basic characteristics of reversible and irreversible structural changes in cells were described; these were later confirmed by a number of studies on various mammalian models and in immediate autopsies on human patients suffering from shock and trauma (Trump et al., '75b). Many questions remain to be answered utilizing the system. These include the specific significance of ion shifts in and between subcellular compartments such as the mitochondria and the endoplasmic reticulum and their relationship to altered structure and function. Elucidation of these requires techniques not previously available such as X-ray microprobe analysis on ultra-thin frozen sections. Currently available techniques promise to make studies possible. Other needed studies include the role of microfilaments and microtubules in the cell membrane function in-
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BENJAMIN F. TRUMP AND RAYMOND T. JONES
STRUCTURE AND FUNCTION OF FISH NEPHRONS
volved in active transport and the role of sodium and potassium movements in organic anion transport. Active trunsport a n d cell structure The studies reported here illustrate that there is a fundamental correlation between ultrastructure and active transport. Agents or conditions resulting i n complete inhibition of active transport generally lead to massive ultrastructural disruption leading to cell death. These do not appear a t random but proceed through a series of regular stages which have been defined. Important in the progression of changes are changes in intracellular organelle compartments as a function of time presumably reflecting ion and water shifts and in large part probably relating to changes in both membrane permeability and active transport. In the late, irreversible stages, irrevocable changes i n membrane permeability apparently occur leading to massive swelling of all cytoplasmic compartments. During the reversible phase one of the more striking changes is condensation of mitochondria, possibly reflecting ion and water flux from that compartment. Pathogenetic sequence of cellular eoents The current results correlating ultrastructure and active transport suggest that two principal types of injury or interaction are associated with both inhibition of transport and loss of cell viability. These are: (1) interference with cell membrane integrity -either by modification of ion pumps or direct modification of protein or lipid components; ( 2 ) interference with mitochondrial ATP synthesis. Both of these events rapidly lead to loss of viability, progressive cell change, and finally, necrosis. Interference with macromolecular synthesis does not appear to result i n either type of change. These findings strongly suggest that complex lethal injuries such as ischemia or chemical toxins operating on Fig. 13 Isolated hogchoker tubule from the second brush border segment incubated in fluorobiphenylacetamide for 60 minutes a t a concentration of 1 0 - 3 M . This renal carcinogen, potent i n rats, i s associated with inhibition of dye transport a n d beginning mitochondria1 changes such as the slight condensations, doughnut and horseshoe forms shown here. Other organelles, including the nucleus, are not altered a t this time. One large multivesicular body (MVB) is present. X 7,500.
38 1
cells in more complex in vivo systems also act through one or the other or both of these pathways. Studies are currently underway in various in vivo and in vitro model systems to determine which factors are of paramount importance. In vitro testing of mutagens and carcinogens Because of the increasing importance in the ecology of mutagenic and carcinogenic agents, tests andlor assay systems are needed to more rapidly assess the long range effects of these compounds. Specifically, the use of in vitro models has proven to be promising in mammalian systems. Similar studies need to be performed in fish and other aquatic animals in order to develop efficient means for testing the deleterious effects on the ecosystem. The isolated flounder tubule model represents a n attempt in this direction which we are employing. Preliminary results presented here reveal that long-term culture with preserved viability and differentiation of these structures is possible. We have reported early cytologic effects of one carcinogen which is capable of producing extensive renal adenocarcinoma in the rat. Early effects on the isolated flounder tubule system are reported here. It is hoped that continuation of such studies in the future will lead to further refinements and improved extrapolation from in vitro to in vivo effects. ACKNOWLEDGMENTS
We wish to thank Ruth E. Bulger (University of Massachusetts) for collaboration and continuing contribution to the field of renal pathophysiology. We also wish to thank the following for their contributions to our studies: Somphong Sahaphong (Mahidol University), Judy M. Strum (University of Maryland), John D. Shelburne (Duke University), Fred L. Ginn (Maine Medical Center) and Jessie Calder and Mary Smith for their technical assistance. Cleveland P. Hickman, Jr. (Washington and Lee University) and William B. Kinter (Mount Desert Island Biological Laboratory) provided valuable contributions to the early phases of the studies. We are extremely grateful to Roy P. Forster (Dartmouth College) for providing the stimulus for our studies of fish nephrons. We are grateful to Connie
382
BENJAMIN F. TRUMP AND RAYMOND T. JONES
Kouns and Elaine Hassman for their help in the preparation of the manuscript and to Charles Moore of The Benidect Research Laboratory for providing the hogchokers. This study was supported in part by NIH grant number AM 15440 and is contribution number 372 from the Comparative and Environmental Pathobiology Program of The Department of Pathology, University of Maryland School of Medicine. LITERATURE CITED Barrett, L. A,, E. M. McDowell, A. L. Frank, C. C. Harris and B. F. Trump 1976 Long-term organ culture of human bronchial epithelium. Cancer Res., 36: 1003-1010. Bulger, R., and B. F. Trump 1968 Renal morphology of the English sole ( P n r o p h e y s u e t z i h s ) . Am. J. Anat., 123: 195-225. 1969a Ultrastructure of granulated arteriolar cells (juxtaglomerular cells) in kidney of a fresh and a salt water teleost. Am. J . Anat., 124: 77-87. 1969b Ca++and K+ ion effects on ultrastructure of isolated flounder kidney tubules. J . Ultrastruct. Res., 28: 301319. 1969c A mechanism for rapid transport of colloidal particles by flounder renal epithelium. J. Morphol., 127: 205-223. Burg, M. B., and P. F. Weller 1969 Iodopyracet transport by isolated perfused flounder proximal renal tubules. Am. J. Physiol., 21 7: 1053-1056. Forster, R. P. 1948 Use of thin kidney slices and isolated renal tubules for direct study of cellular transport kinetics. Science, 188: 65-67. 1953 A comparative study of renal function in marine teleosts. J. Cell Comp. Physiol., 42: 487-510. 1961 Kidney cells. In: The Cell. J. Brachet and A. E. Mirsky, eds. Academic Press, New York, Vol. 5, pp. 89-16], 1967 Renal transport mechanisms. Fed. ROC.,26: 1008-1019. Forster, R. P., and F. Berglund 1956 Osmotic diuresis and its effect on total electrolyte distribution in plasma and urine of the aglomerular teleost, Lophizrs nmericnniis. J. Gen. Physiol., 39; 349-359. 1957 Contrasting inhibitory effects of probenecid on the renal tubular excretion of PAH and on the active reabsorption of urea in the dogfish, Sqiinlus ncnnthins. J. Cell. Comp. Physiol., 49: 281-285. Forster, R. P., and S. K. Hong 1958 I ~ znitro transport of dyes by isolated renal tubules of the flounder as disclosed by direct visualization. Intercellular accumulation and transcellular movement. J. Cell. Comp. Physiol., 51: 259-272. Forster, R. P., I. Sperber and J. V. Taggart 1954 Transport of phenolsulfonphthalein dyes in isolated tubules of the flounder and i n kidney slices of the dogfish. Comparative phenomena. J. Cell. Comp. Physiol., 44: 315-318. Hickman, C. P., and B. F. Trump 1969 The kidney. In: Fish Physiology. W. S. Hoar and D. J. Randall, eds. Academic Press, New York, Vol 1, pp. 91-239. Enter, W. B. 1966 Chlorophenol red influx and
eflux: microspectrometry of flounder kidney tubules. Am. J. Physiol., 211: 1152-1164. Puck, T. T., K. Wasserman and A. P. Fishman 1952 Some effects of inorganic ions on the active transport of phenol red by isolated kidney tubules of the flounder. J. Cell. a m p . Physiol., 40: 73-88. Sahaphong, S., and B. F. Trump 1971 Studies of cellular injury i n isolated kidney tubules of the flounder. V. Effects of inhibiting sulfhydryl groups of plasma membrane with the organic mercurials PCMB (parachloromercuribenzoate) and PCMBS (parachloromercuribenzenesulfonate). Am. J. Pathol., 63: 277-298. Shelburne, 3. D., A. U . Arstila and B. F. Trump 1973 Studies on cellular autophagocytosis. The relationship of autophagocytosis to protein synthesis and to energy metabolism in rat liver and flounder kidney tubules in uitro. Am. J. Pathol., 73: 641-4570. Trump, B. F., and A. U. Arstila 1975 Cellular reaction to injury. In: Principles of Pathobiology. M. F. LaVia and R. B. Hill, eds. Oxford University Press, New York, Second edition, pp. 9-96. Trump, B. F., and R. E. Bulger 1967 Studies of cellular injury in isolated flounder tubules. I. Correlation between morphology and function of control tubules and observations of autophagocytosis and mechanical cell damage. Lab. Invest., 16: 453-482. 1968a Studies of cellular injury in isolated flounder tubules. 111. Light microscopic and functional changes due to cyanide. Lab. Invest., 18: 721-730. 1968b Studies of cellular injury i n isolated flounder tubules. IV. Electron microscopic observations of changes during the phase of altered homeostasis in tubules treated with cyanide. Lab. Invest., 18: 731-739. 1971 Experimental modification of lateral and basilar plasma membranes and extracellular compartments in the flounder nephron. Fed. Proc., 30: 22-41. Trump, B. F., and F. L. Ginn 1968 Studies of cellular injury i n isolated flounder tubules. 11. Cellular swelling in high potassium media. Lab. Invest., 18: 341-351. 1969 The pathogenesis of subcellular reaction to lethal injury. In: Methods and Achievements in Experimental Pathology. E. Bajusz and G. Jasmin, eds. Yearbook Chicago, VO~. 4, pp. 1-29. Trump, B. F., R. T. Jones and S. Sahaphong 1975a Cellular effects of mercury on fish kidney tubules. In: The Pathology of Fishes. W. E. Ribelin and G. Migaki, eds. University of Wisconsin Press, Madison, pp. 585-612. Trump, B. F., J. M. Valigorsky, R. T. Jones, W. J. Mergner, J. H. Garcia and R. A. Cowley 1975b The application of electron microscopy and cellular biochemistry to the autopsy: Observations on cellular changes in human shock. Human Pathol., 6: 499-516. Trump, B. F., W. J. Mergner, M. W. Kahng and A. J. Saladino 1976 Studies on the subcellular pathophysiology of ischemia. Circulation, 53: Suppl. I, 17-26. Wasserman, K., E. L. Becker and A. P. Fishman 1953 Transport of phenol red in the flounder renal tubule. J. Cell. Comp. Physiol., 42: 385393.
ABSTRACT In the present study we have extended our investigations concerning the correlation between ultrastructure and active transport in the isolated flounder nephron. The composition of the fish nephron is defined in ultrastructural terms and its behavior when incubated in vitro under short term and long term culture conditions is described. Using the in vitro system originally described by Forster, a variety of inhibitors and conditions which modify cell structure and function were tested. Ultrastructure was correlated with chlorphenol red dye transport. In general, conditions altering active transport also markedly altered cellular ultrastructure. The principal alterations consisted of membrane changes involving various organelles -most importantly the plasma membrane and the mitochondria. Conditions associated with irreversible cell injury could be rapidly produced by interference either with mitochondrial ATP synthesis or with the integrity of the plasma membrane. Both of these rapidly lead to irreversible events which are preceded by reversible structural changes. Organelle changes progress in a rather well-defined sequence of reversible and irreversible stages which are defined. One difference between the two types of interactions is the presence of intramitochondrial calcification which does not occur with direct modification of the mitochondrial electron transport system. The concept of utilizing long term explant organ cultures of fish nephrons for environmental studies is introduced.
The purpose of this paper is to present a synthesis of studies performed in our laboratory on the relationship between ultrastructure and active transport i n the fish nephron. We will attempt to correlate the effects of a variety of extrinsically induced interactions on structure and active transport in the teleost nephron-briefly touching on some that have been reported previously yet expanding this to conditions not heretofore described and finally attempting a synthesis of this information into an integrated picture of the subject. The history of active transport studies in the fish nephron spans several decades and has enriched the general biology of transport in general and of fish and mammalian nephron transport in particular. Studies on the system reported here were first introduced by Forster ('48) i n his paper on the use of isolated nephrons and thin kidney slices for studying active transport. In a brilliant series of studies, Forster and his colleages ('53, '54, '56, '57, '58, J. EXP. ZOOL.,199: 365-382
'61, '67) systematically explored many of the characteristics of the transport systems and determined many of their metabolic requirements. These studies, supplemented by those of Puck et al. ('52) and Wasserman et al. ('53) developed the concept of a two-stage system, one involving entry from the suspending medium into the cells and the second, from the cells into the tubule lumen. Later Kinter ('66) using microspectrophotometry and radioactively labelled chlorphenol. red began to develop more quantitative estimates and kinetic analyses of transport in these systems and Burg and Weller ('69) using isolated perfused tubules further characterized the process. In our laboratory we began i n the late fifties to develop a n ultrastructural understanding of the nephron and to attempt correlation between changes in ultrastructure and the active transport process (see review by Trump and Bulger, '71). As a spin-off of that work we began to define the regional characteristics of the fish 365
366
BENJAMIN F. TRUMP AND RAYMOND T. JONES
nephron and several years ago, together with Hickman (Hickman and Trump, '69) attempted to integrate all available morphologic and functional information on the kidneys of fish of all types. All of these fish studies have had many implications for biomedical science. They have been of obvious direct importance to the understanding of fish physiology and have contributed substantially to the elucidation of renal structure and function in the human. The studies have also contributed substantially to the general cell biology of active transport and moreover have made essential contributions to the development of modern cellular pathology, since cellular reaction to injury as it is now known, was in large part developed by the study of these isolated fish nephrons with which controlled experimentation in vitro could be performed (Trump and Ginn, '69). Most of the conclusions derived from earlier studies have stood us in good stead in subsequent studies on mammalian disease at the ultrastructural level including human disease. MATERIALS AND METHODS
Specimens of three species of flatfish were utilized in these studies. All were obtained by either dragging or gill netting and maintained in sea water aquaria or artificial sea water aquaria at approximately 14" C. Species utilized were Porophrys uetulus, the English sole from Puget Sound near Seattle, Washington; Paralichthys lethostigma, the Southern flounder from the estuaries near Beaufort, North Carolina; and Trinectes maculatus, the hogchoker, from the Potomac River near Morgantown, Maryland and the Patuxent River near Benedict, Maryland. In all cases the preparations were obtained by sacrificing the fish quickly, removing the kidney and immersing it in cold (0-4" C) Forster's buffer with the following composition in mMoles per liter; NaCl, 135; KC1, 2.5; CaC12, 1.5; MgC12, 1.0; NaH2P04, 0.5; NaHC03, 10. Small fragments measuring a few mm in diameter were quickly cut with scissors and these were, in turn, teased with dissecting needles into separated clumps of tubules. These were maintained in Forster's buffer at 0-4" C until used. Two types of incubation were carried
out. For short term incubation the tubules were incubated in suspensions in Petri dishes or small centrifuge tubes and gassed by bubbling oxygen through the medium via a 21 gauge hypodermic needle. For long term incubations the tubules were placed in 60 mm plastic Petri dishes containing CMRL 1066 with 1 pg insulin per ml, 0.1 pg hydrocortisone hemisuccinate per ml, 2 mM L-glutamine, 300 U penicillin G per ml, 300 pg streptomycin per ml, and 2.5 pg Amphotericin B/ml, 5 % heat-inactivated fetal calf serum. The dishes were maintained in a controlled atmosphere chamber and gassed with a mixture of 45% 02,50% N2 and 5 % COz. The chamber was rocked at 10 times per minute and maintained at 37" C. Transport was studied by incubation in media containing 1 X 10-4 molar chlorphenol red and by periodic observation of the tubule clumps by light microscopy. The transport was noted as intraluminal accumulation which was scored as follows: 0 , no accumulation; 1 coloration just detectable; 2 , intermediate coloration; and 3 , intense coloration. Tissues were fixed for light and electron microscopy in either 1% osmium tetroxide buffered with s-collidine, 4 % glutaraldehyde, or a mixture of 4 % formaldehyde and 1 % glutaraldehyde in 0.2 molar phosphate buffer. The aldehyde fixed tissues were washed and post-fixed in osmium tetroxide. All tissues were dehydrated in ethanol and embedded in Epon. Thin sections were cut and double-stained with uranyl magnesium acetate and lead citrate. Compounds were added directly to the incubation media in the final concentrations noted in the results and in the table. In case of lipid soluble inhibitors these were dissolved in a small amount of ethanol. Controls were run containing similar amounts ofethanol.
+
+
+,
Fig. 1 Electron micrograph of hogchoker kidney tubule from the second brush border segment incubated in oxygenated Forster's buffer for 1 hour. Note the excellent preservation of cellular structure. The lumen (L) is at the top; the basement membrane (BM) is a t the lower left. Note the appearance of nucleus, mitochondria (M), basilar infoldings of cell membrane (free arrow), apical junctional complexes (JC) and intracellular membranes including endoplasmic reticulum. A small amount of cell debris is in the lumen at the top. Incubated flounder nephrons maintain this type of ultrastructure for many hours. x 5,000.
STRUCTURE A N D FUNCTION OF FISH NEPHRONS
367
368
BENJAMIN F. TRUMP AND RAYMOND T. JONES RESULTS
The marine teleost nephron The nephron of the typical marine teleost appears to consist of the following segments beginning after the renal corpuscle (Bulger and Trump, '68): a neck segment, a first proximal segment, a second proximal segment which sometimes has a terminal third segment, a collecting tubule, and a collecting duct. A typical distal segment resembling that in mammals is not seen in the marine teleosts thus far studied in detail; nor do they possess a ciliated intermediate segment between the second or third proximal segment and the distal or collecting tubule. The following is a description of the typical features of the several segments. A renal corpuscle with juxtaglomerular cells is typically present (Bulger and Trump, '69a). The neck segment is made up of ciliated cuboidal cells that contain many mucous granules. The next segment of the nephron is the first proximal segment (PSI). This segment is composed of columnar cells with a well-developed cytocavity network. Numerous large lysosomes as well as large mitochondria are present. Closely packed microvilli along with apical tubules at their base are further identifying features of this segment. The cells of proximal segment I1 are similar to those of PSI, however, they are somewhat taller and the microvilli are sparser. Mitochondria are scattered throughout the cell and the basilar membranes form a well-developedlabyrinth. The third proximal segment, when present, consists of similar, although shorter, cells with an abundant endoplasmic reticulum (ER). The collecting tubule cells also have apical mucous granules, but only a few microvilli and an occasional cilium. The collecting tubules converge into a collecting duct system whose cells are surrounded by smooth muscle cells (Hickman and Trump, '69; Trump and Bulger, '71). Control tubules in uitro; Short-term When control tubules are incubated in Forster's buffer and gassed with oxygen, active transport proceeds briskly with marked intraluminal accumulation as previously noted by Forster and good preservation of ultrastructure (Trump and Bulger, '67). Transport appears to occur in all proximal segments. It seems unlikely that the neck transports but it is difficult to
state this with certainty. It does not appear that transport occurs in the collecting ducts. Morphologically, the tubules, which are largely collapsed at the time of removal from the fish with blebs of cytoplasm into the lumen, inflate during the incubation and maintain good preservations for more than 8 hours (fig. 1). Using these incubation conditions there is good preservation of mitochondria, cell membranes, nucleus, endoplasmic reticulum, Golgi apparatus and lysosomes throughout the period. The only significant ultrastructural changes that occur are the formation of numbers of lysosomes containing degenerative organelles such as mitochondria. Many of these appear to result from autophagocytosis, as previously described, (Trump and Bulger, '67) involving sequestration of organelles by wrapping with ER cisternae forming autophagic vacuoles (AV). Some of the larger AV may also have contributions from phagocytosis of debris resulting from damaged tubules passing down the lumen and phagocytosis by the epithelium. It is apparent that this increased organelle turnover and accumulation of AV is not associated with impairment of active transport. It is also not clear whether the accumulation of AV results from an increased rate of formation in incubated tubules or from a decreased rate of turnover andlor extrusion of contents from these bodies. Also, systems for rapid transport of colloidal particles from lumen to both exist (Bulger and Trump, '6%).
Control tubules in uitro; Long term When the tubules are cultured under the organ culture conditions previously characterized for mammalian bronchi and pancreatic duct (Barrett et al., '76) good viability can be maintained for as long as Fig. 2 Electron micrograph of hogchoker kidney culture maintained i n CMRL 1066 medium for 15 days. The lumen (L) is at the top, the basement membrane area toward the lower right. The lack of a well-defined basement membrane in this area may indicate that this is one of the outgrowths of tubular structures that occur at the margins of these explant cultures. Note the appearance of the cell which is compatible with continued viability. However, certain changes can be clearly identified such a s the marked shape change i n the mitochondria which are frequently bowlor doughnut-shaped, presence of annulate lamellae (arrow), and numerous residual bodies (RB) or secondary lysosomes presumably derived from extensive autophagocytosis. Note preservation of junctional complexes along the lumen. X 5,000.
STRUCTURE AND FUNCTION OF FISH NEPHRONS
369
TABLE 1
w e c t s of various c o m p o u n d s and altered conditions on active transport Condition
Conc.
LET to Zero
Condition
Conc.
LET to Zero
+
~
Mitochondrial znhibztors CN 3 x 10-3 Antimycin 2x
Azide Oligomycin BAL
Tvrocidine 2,4 DNP
10-3 10-4 10-4 10-5 10-2 10-3 10-4 10-5 10-2 10-3 10-4 10-5 10-3 10-4 10-3 10-4 10-5
Continuous 15 min 30 min 1 hr 2 hr 4 hr
N2
Amytal
10-3 10-4 10-5
Glycolytic inhibitors IAA
KF
10-3 10-4 10-5 10-3 10-4 10-5
Membrane active c o m p o u n d s Ouabain 10-2 10-3 10-4 10-5 10-3 10-4 10-5 12.5 ygtml 1.25 yglml
Gramicidin Amphotericin
.1mglml
Filipin
.01 mg/ml 10 mg%
Vitamin A
20 mg% 2 gm%
Saponin Digitonin
10-4 10-5
CPC
10-3 10-4 5 x 10-4
Chlorcquine
5 x10-5
5 x 10-6
Phospholipase
.2 mglml .02 mglml
Modification of m e d i u m Mg-free Ca free EDTA K-free PH-3 pH-9.3
24 hr 30 min-0 30 min-0 30 min-0 15 min-0 4 hr-0 2 hr-0 No effect 2 hr-k 6 hr-? 30 min-0 30 min-0 Minimal effect No effect 30 min-0 Minimal 15 min-0 15 min-0 Minimal effect 2 hr-1 No effect No effect 1hr-2+ 2 hr-2 4 hr-0 0 0-s1igh t Very slight
+
+
15 min-0
2 hr-0 No effect No effect No effect 1hr-0 30 min-0
4 hr-0 4 hr-0 4 hr-2 No effect No effect 4 hr-0 4 hr-O-l+ 2 hr-0 2 hr-0 No Inhib. 6 hr-+ 15 min-0 4 hr-0 6 hr-0 5 min-0 5 min-0 4 hr-0 No effect No effect 15 min-0 1 hr-0
+
+
20 c 5 hr
10 - 2 1.8 X 10-2 3.5 X 10-2 6.9 X 10-2 1.4 X 10-1
+
Hypotonia .75 Forster's .5 Forster's
Normal Normal 30 min-0 5 min-0
'25 Forster's Dist. HzO
Heauy m e t a l s Arsenate Arsenite PCMB
10-2 10-3 10-4 10 - 2 10-3 10-4 10-2 10-3 10-4 10-5
PCMBS
10-3 10-4 10-5
Hgaz
10-3
1hr-0
CHTHgCI
CdS04
10-4 10-5 10-6 10-3 10-4 10-5 10-6 10-3
10-4
Pb citrate ZnClZ
10-5 10-6 10-7 10-4
_7 _0 - 5
10-6 10-3 10-4 10-5
Inhibitors of macromolecular synthesis Puromycin 2 x 10-4 Pactamycin 10-4 10-5 10-6
Actinomycin
50 yg/ml 5 yglm1 10-4
2 hr-0 4 hr-0 4 hr-0 15 min-0 2 hr-+ Minimal 15 min-0 15 min-0 2 hr-0 4 hr-+ 15 min-0 2 hr-0 4 hr-slight effec 15 min-0 15 min-0 4 hr-1 6 hr-2+ 15 min-0 15 m i n d 6 hr-slight effec 6 hr-no effect 15 min-0 15 min+J 15 min-0 1hr-0 4 hr-1 Minimal No effect No effect 15 min-0 Minimal Minimal
+
+
6 hr-No effect No effect No effect No effect 5 hr-+
30 min-+ Cells 3 1 hr-0 No cell or lumen 15 min-0
Chloramphenicol 20 yglml 2 m/ml
No effect No effect No effect 4 hr-+ No effect 4 hr-+ No change
p-fluorophenylalanine
5 hr-0
1 hr-0
Carcinogens fluoro-biphenylacetamide
+
30 min-0
PH-10.5
48 h r 3 days 4 days KCl
Cells-1 4 hr-0 1 hr-0 1hr-0 1 hr-0 1 hr-2 1 hr-+ 2 hr-0 1hr-0-+ 1hr-0
No effect
Streptovitacin
3 X 10-6 Cycloheximide
Ethionine
10-4 10-5
400 yglml 40 yglml 10-2 10-3
10-3 M
5 hr-+ 1 hr-0 1hr-0
Slight inhib. by 1h r
LET, least effective time; BAL, antilewisite; IAA, Iodoacetate acid; KF, Potassium flvoride; CPC, Cetyl... - British . ~
STRUCTURE AND FUNCTION OF FISH NEPHRONS
371
Fig. 3 Basilar portion of tubular epithelial cells of Southern flounder after 4 hours incubation with 1 0 - 4 antimycin. This treatment is associated with complete inhibition of electron transport and chlorophenol red dye transport. The cells undergo early swelling and later become irreversibly altered. Such a stage is shown here. Note the marked swelling of mitochondria (M) with tubular conversions of cristae, lamellar stacks of basilar infoldings (free arrow) and a nucleus (N) showing karyolysis. The endoplasmic reticulum (ER) is dilated. X 5,000. Fig. 4 Isolated Southern flounder kidney tubule incubated 30 minutes with potassium cyanide in the presence of added ATP. ?tyo types of cell change are shown. In the first, shown on the left, the mitochondria are condensed with expansion of the outer compartment, while i n the cell on the right mitochondria show high amplitude swelling. Both of these changes are reversible and the condensed conformation appears to occur earlier i n time as indicated i n the cell stages mentioned in the text. X 15,000.
two weeks, although during this time the tubules maintained an altered form and the ultrastructural findings were associated with good viability (fig. 2). At the edges of the cultured fragments apparent division and growth of cells occurs regenerating a layer of epithelium around the periphery of the clump which in areas begins to form intercellular canaliculi with cell junctions that were suggestive of attempted lumen formation. Cells in these prolonged cultures retained a polarity with junctional complexes toward the luminal surface but alterations occurred within the cytoplasm. Mitochondria changed from their normally elongate configuration to rounded doughnut-shaped profiles often associated with the close apposition of lipid droplets. Close packing of cristae and lamellar formations between cristae were also observed.
Inhibition of Oxidative metabolism
In the table is shown the effects of a variety of inhibitors of mitochondria1 function including inhibitors of the electron transport system, inhibitors of oxidative phosphorylation, and uncouplers of oxidative phosphorylation. M e n these compounds were tested, at the concentrations indicated, on isolated transporting tubules, inhibition of active transport was noted fairly rapidly in all cases. The molar concentrations required for maximum inhibition varied as indicated. In general, the inhibition was initially reversible in the sense that when the tubules were removed from the inhibitor-containing medium and replaced into normal medium with oxygenation, transport was again reinstituted. This phase lasted from 30 minutes to 2
3 72
BENJAMIN F. TRUMP AND RAYMOND T. JONES
STRUCTURE AND FUNCTION OF FISH NEPHRONS
373
Fig. 6 Isolated English sole tubule incubated 6 hours i n medium containing 1 0 - 4 M 2,4-dinitrophenol. This is a first brush border segment showing numerous lysosomes characteristic of this region, but showing irregularities and slight condensation of mitochondria typical of early mitochondrial changes following uncoupling or inhibition of respiration. X 5,000.
hours. The most useful condition for studying this type of reversibility was bubbling with purified nitrogen since in that case one is not concerned with removal of residual toxic compounds even in the replacement medium. During the course of incubation, light microscopy using phase or Normarski optics revealed that the cells consistently underwent softening of contours, swelling, and obliteration of lumens. With careful studies at the longer time intervals it could be seen that all mitochondria within the Fig. 5 Tubule from the first brush border segment of a n English sole incubated for 1 hour in medium gassed with purified nitrogen. This was associated with inhibition of dye transport which was reversible at this stage. Note the mild clumping of chromatin (Ch), and tiny flocculent densities (free arrow) i n the mitochondria. Both of these are known to be reversible in this system and subsequently have been found to be reversible alterations in mammalian systems as well. X 6,500.
epithelial cells had undergone swelling to form a population of spherical profiles in the cvtotdasm. Election microscopic studies of the progression of change following such inhibitors revealed a fairly reproducible sequence of stages through which the cells pass between the beginning or 0-time state and the final irreversible or necrotic state (fig. 3 ) (Trump and Bulger, '68a,b). Furthermore, by comparing these stages with the point of loss of reversibility the reversible ones (figs. 4 and 5) could be distinguished from those which were irreversible. For purposes of the discussion we have arbitrarily numbered these stages from 1 , the normal cell, to 7, the advanced necrotic cell (Trump and Ginn, '68; Trump et al., '76). In stage l a the nuclear chromatin is clumped and the mitochondrial granules disappear along with a reduction in cell glycogen. Cells of stage 2 of cell injury
374
BENJAMIN F. TRUMP AND RAYMOND T. JONES
STRUCTURE AND FUNCTION OF FISH NEPHRONS
have dilated ER, distorted plasma membranes and slightly swollen lysosomes. Stage 3 is characterized by condensed mitochondria and expanded cell sap (fig. 4) while in stage 4 some of the mitochondria are swollen while others are condensed. The mitochondria are all swollen in stage 4a and there is some loss of the polysome arrangement. In stage 4b, tiny flocculent densities are present in the mitochondria. Flocculent mitochondrial densities are the hallmark of stage 5 . Stage 5a is similar to stage 5 ; however, mitochondrial calcifications are present. Lysosomes have lost their integrity in stage 6 while in 6a there are mitochondrial calcifications. As mentioned above, stage 7 represents the completely necrotic cell in which large myelin forms have formed along with karyolysis and fragmented cell membranes. Stage 7a has mitochondrial calcifications. Stages 1 through 4 b of cell injury appear to be reversible while stages 5 through 7a appear to be irreversible (Trump and Arstila, '75). The changes indicated above in stages 2 through 7a are cumulative. It is noteworthy that in none of these conditions did mitochondrial accumulations of calcium phosphate occur (fig. 6). This is in contradistinction to some of the other conditions noted below. The difference is apparently the lack of active mitochondrial calcium uptake in the presence of a potent mitochondrial inhibitor or uncoupler.
375
tively little effect was noted. This may be due to the fact that ATP can be formed in the presence of fluoride and also because other substrates such as fatty acids may be utilized in addition to those arising from the glycolytic sequence. Since prominent glycogen deposits are not present in flounder nephrons, the latter seems especially likely. Iodoacetate had very severe effects similar to those of inhibition of oxidative metabolism above; however, it may well be that the effects are due to changes other than in the glycolytic sequence such as the effect of iodoacetate on membrane sulfhydryl groups.
Cell membrane interactions Over a fairly wide series of concentrations ouabain treatment was associated with inhibition of dye transport and cell swelling with direct reproduction of the phases described under oxidative inhibition above. A very important difference was that in this case mitochondrial calcification did occur (fig. 7). It was further noted that respiration persisted in the presence of ouabain. The cell swelling was often extremely marked.
PCMB and PCMBS The organic mercurials also resulted in marked inhibition of transport and also in rapid cell swelling and necrosis (over a fairly wide range of concentrations) (Sahaphong and Trump, '71). The fact that changes were similar with PCMBS (paraGlycolytic inhibitors chloromercuribenzenesulfonate), a relativeMixed results were obtained with these ly non-penetrating mercurial and PCMB compounds. With fluoride at fairly high (parachloromercuribenzoate), suggested concentrations as shown in the table, rela- that the most important interaction was involved at the surface membranes. We also Fig. 7 Southern flounder kidney tubule from the second brush border region incubated in the noted a potential reversibility of the transpresence of 1 0 - 3 M ouabain for 2 hours. Note that port defect with the PCMB and PCMBS as most mitochondria are swollen while one is con- observed with the oxidative inhibitors (durdensed (arrow). The swollen mitochondria contain ing the first few minutes). Also, as with densities (D) adjacent to the inner membrane and membranes of the cristae which can be shown to the ouabain, mitochondrial calcification represent intramitochondrial calcium phosphate was seen except with the highest concenaccumulations. This cell is probably irreversibly trations. This again is presumed to indialtered. Note dilatation of ER and enlarged, swol- cate lack of penetration of the mercurial len cell sap. X 10,000. into the cells since isolated mitochondria Fig. 8 Southern flounder tubule incubated in the presence of amphotericin (1.3 mglml) for 6 incubated with PCMB showed marked inhours. This treatment is associated with inhibition hibition of calcium uptake (Trump et al., of dye transport and swelling of some mitochondria '75a). Both HgClz and CH3-HgCl2had simiand condensation of others. Note the large intracytoplasmic vacuoles and obliteration of basilar lar, even more rapid effects on both transport and ultrastructure. membrane conformations. X 5,000.
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Other agents
Treatment of isolated tubules with ionophores such as amphotericin and filipin also resulted in rapid cytolysis (fig. 8). This was preceded by condensation of mitochondria similar to that which was observed previously with amphotericin treatment of toad bladder. Direct treatment with various surface active agents including saponin, digitonin, cetylpyridinium chloride, and phospholipase all resulted in marked inhibition. With surface saponin (fig. 9), direct dissolution of basilar cell membranes could be noted. Modification of the m e d i u m Elimination of magnesium from the medium was associated with marked retardation of transport with inhibition complete by 1 hour. Electron micrographs showed first condensation and then later swelling of mitochondria. Ultimately, the pattern was similar to that observed with the oxidative inhibitors. Incubation in calciumfree medium or calcium-free medium in the presence of EDTA produced marked alterations of both structure and transport (Bulger and Trump, '69b). With time cells began to separate and soon even by light microscopy the pressure of a cover-slip on the preparation caused extrusion of cells from the tubules leaving the basement membrane cylinders intact. By electron microscopic study complete dissociation of all types of junctions including tight, intermediate, desmosomes and gap junctions was noted. Large channels then existed between the lumen and the basement membrane. In addition, there was marked disorganization of intracellular organelles with complete disorientation of cell apices. Fig. 9 Tubule from Southern flounder incubated in the presence of saponin for 4 hours. Note dissolution of basilar infoldings (free arrow) and marked swelling of mitochondria (M) and other membrane systems such a s the ER. Organelles are difficult to recognize. There is virtually immediate cessation of active transport. Basement membrane, BM. x 8,000. Fig. 10 Isolated tubule from second brush border segment of English sole incubated i n medium devoid of potassium for 30 minutes. This is associated with neither intracellular nor intraluminal accumulation. Note cell sap swelling and marked change of basilar infoldings, some of which (free arrows) appear to have pulled away completely from the basilar cell membrane. x 10,000.
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The cells tended to undergo enspherulation. Half-desmosomes could be observed along lateral cell margins. No intraluminal dye accumulations could be noted under these conditions although intracellular accumulation still occurred at least during the early stages. Incubation of tubules in potassium-free medium results in neither intraluminal nor intracellular accumulation. There was, however, some cell swelling with marked distortion of basilar cell membranes leaving large basilar compartments with only a relatively simple plasma membrane (fig. 10) (Bulger and Trump, '69b). Replacement of sodium with potassium chloride had the following effects. At 10 and 18 mM potassium there was only slight inhibition of dye transport. At 35, 69 and 140 mM potassium there was marked inhibition complete by 1 or 2 hours. Furthermore, at these latter three concentrations there was marked cell swelling. This was preceded by a phase in which mitochondria were condensed but ER and cell sap were dilated. The condition reproduced the stages described above (Trump and Ginn, '68). Modification of Forster's buffer to produce hypotonic stress and study of volume regulation had the following results. At three-fourths strength Forster's buffer adaptation appeared to occur with both normal morphology and normal dye transport. At one-half strength Forster's buffer, transport was almost normal and many normal tubules occurred for a time. However, at either one-fourth strength Forster's buffer or in straight distilled water no dye transport and marked swelling and cytolysis occurred (fig. 11). At the ultrastructural level the cells rapidly progressed to marked high amplitude mitochondrial swelling. Reduction to pH 3 led to rapid inhibition of transport within the first few minutes. Among the striking ultrastructural effects noted was marked clumping of nuclear chromatin. Elevation of pH to 9.3 or 10.5 also led to inhibition of transport. The main difference at the elevated pH was the pronounced cell swelling that occurred very early and rapid progression to stage 7. Incubation of tubules at 0-4" C followed by reversibly testing led to the observation that cells could tolerate 5 hours of
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cold incubation with no effect on structure and function. By 24 hours there was retention of transport however with incubations of 2 to 4 hours, transport inhibition was seen. At 3-7 days of cold incubation no transport was seen by 1 hour. Electron micrographs of cells incubated at reduced temperature showed a series of stages comparable to those observed with oxidative inhibition though the rate of change was much slower (fig. 12). Heavy metals A number of heavy metals including arsenate, mercury, cadmium, lead, and zinc have been tested. All of these at some concentration exhibited complete inhibition of dye transport. Mercury and cadmium were among the most toxic of these heavy metals and led rapidly to irreversible cell changes. We are presently comparing the effects of heavy metals on the entire fish as well as on the in vitro tubules. Inhibitors of macromolecular synthesis A variety of inhibitors of macromolecular synthesis have been tested as shown in the table. Although some of these at very high concentrations had slight effects on transport, most had no effect. At the ultrastructural level most changes were compatible with cell survival though some, such as actinomycin, altered polysome and nucleolar morphology (Shelburne et al., '73). Effects of carcinogenic agents So far we have just begun studies of carcinogens in systems maintained for relatively long periods of time in vitro. Our initial studies are with the organic carcinogen fluorobiphenyl acetamide which is known to consistently produce renal adenoFig. 11 Isolated Southern flounder kidney tubule incubated in one-fourth strength Forster's buffer for 30 minutes. Under these conditions cell swelling occurs rapidly with obliteration of basilar processes, enlargement of cell sap, swelling of most mitochondria and dilatation of ER. Some basilar infoldings persist. Volume regulation cannot occur i n this osmolality. Cell swelling and death occur rapidly. X 6,000. Fig. 12 Southern flounder tubule incubated i n Forster's buffer at 0-4" C for 1 week. Under these conditions ability for recovery is lost after approximately 1-2 days. By 1 week irreversible stage 5 changes, including swollen cell sap, swollen mitochondria, flocculent densities and fragmented ER can be seen. X 5,000.
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carcinomas in the rat following long term feeding. This compound, at a concentration of 10-3 M, was associated with inhibition of dye transport although this was never complete. Many tubules showed slight intraluminal accumulation. Ultrastructural studies of the effects of this agent on the tubular epithelium are preliminary but so far indicate a very early effect on tubular cell mitochondria which undergo slight condensation with shape irregularities, formation of ring and doughnut forms, and little other subcellular change. Studies are currently underway testing the longer term effects. DISCUSSION
The unity of the isolated flounder tubule system for the study of cell pathology is shown by the present results as well as those previously published. It is especially suitable for studying the correlation between ultrastructure and active transport as well as for examining a wide variety of cellular reactions to injury. The results of experiments performed in our laboratory over the past 20 years using this system have clearly been of value in determining the relationship between organelle change and cell function and have laid the foundation for further studies on the relationship between ultrastructure and function in injured cells in a variety of higher animal systems including man (Trump and Arstila, '75; Trump et al., '76). Using this system, originally described by Forster ('48), the basic characteristics of reversible and irreversible structural changes in cells were described; these were later confirmed by a number of studies on various mammalian models and in immediate autopsies on human patients suffering from shock and trauma (Trump et al., '75b). Many questions remain to be answered utilizing the system. These include the specific significance of ion shifts in and between subcellular compartments such as the mitochondria and the endoplasmic reticulum and their relationship to altered structure and function. Elucidation of these requires techniques not previously available such as X-ray microprobe analysis on ultra-thin frozen sections. Currently available techniques promise to make studies possible. Other needed studies include the role of microfilaments and microtubules in the cell membrane function in-
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volved in active transport and the role of sodium and potassium movements in organic anion transport. Active trunsport a n d cell structure The studies reported here illustrate that there is a fundamental correlation between ultrastructure and active transport. Agents or conditions resulting i n complete inhibition of active transport generally lead to massive ultrastructural disruption leading to cell death. These do not appear a t random but proceed through a series of regular stages which have been defined. Important in the progression of changes are changes in intracellular organelle compartments as a function of time presumably reflecting ion and water shifts and in large part probably relating to changes in both membrane permeability and active transport. In the late, irreversible stages, irrevocable changes i n membrane permeability apparently occur leading to massive swelling of all cytoplasmic compartments. During the reversible phase one of the more striking changes is condensation of mitochondria, possibly reflecting ion and water flux from that compartment. Pathogenetic sequence of cellular eoents The current results correlating ultrastructure and active transport suggest that two principal types of injury or interaction are associated with both inhibition of transport and loss of cell viability. These are: (1) interference with cell membrane integrity -either by modification of ion pumps or direct modification of protein or lipid components; ( 2 ) interference with mitochondrial ATP synthesis. Both of these events rapidly lead to loss of viability, progressive cell change, and finally, necrosis. Interference with macromolecular synthesis does not appear to result i n either type of change. These findings strongly suggest that complex lethal injuries such as ischemia or chemical toxins operating on Fig. 13 Isolated hogchoker tubule from the second brush border segment incubated in fluorobiphenylacetamide for 60 minutes a t a concentration of 1 0 - 3 M . This renal carcinogen, potent i n rats, i s associated with inhibition of dye transport a n d beginning mitochondria1 changes such as the slight condensations, doughnut and horseshoe forms shown here. Other organelles, including the nucleus, are not altered a t this time. One large multivesicular body (MVB) is present. X 7,500.
38 1
cells in more complex in vivo systems also act through one or the other or both of these pathways. Studies are currently underway in various in vivo and in vitro model systems to determine which factors are of paramount importance. In vitro testing of mutagens and carcinogens Because of the increasing importance in the ecology of mutagenic and carcinogenic agents, tests andlor assay systems are needed to more rapidly assess the long range effects of these compounds. Specifically, the use of in vitro models has proven to be promising in mammalian systems. Similar studies need to be performed in fish and other aquatic animals in order to develop efficient means for testing the deleterious effects on the ecosystem. The isolated flounder tubule model represents a n attempt in this direction which we are employing. Preliminary results presented here reveal that long-term culture with preserved viability and differentiation of these structures is possible. We have reported early cytologic effects of one carcinogen which is capable of producing extensive renal adenocarcinoma in the rat. Early effects on the isolated flounder tubule system are reported here. It is hoped that continuation of such studies in the future will lead to further refinements and improved extrapolation from in vitro to in vivo effects. ACKNOWLEDGMENTS
We wish to thank Ruth E. Bulger (University of Massachusetts) for collaboration and continuing contribution to the field of renal pathophysiology. We also wish to thank the following for their contributions to our studies: Somphong Sahaphong (Mahidol University), Judy M. Strum (University of Maryland), John D. Shelburne (Duke University), Fred L. Ginn (Maine Medical Center) and Jessie Calder and Mary Smith for their technical assistance. Cleveland P. Hickman, Jr. (Washington and Lee University) and William B. Kinter (Mount Desert Island Biological Laboratory) provided valuable contributions to the early phases of the studies. We are extremely grateful to Roy P. Forster (Dartmouth College) for providing the stimulus for our studies of fish nephrons. We are grateful to Connie
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Kouns and Elaine Hassman for their help in the preparation of the manuscript and to Charles Moore of The Benidect Research Laboratory for providing the hogchokers. This study was supported in part by NIH grant number AM 15440 and is contribution number 372 from the Comparative and Environmental Pathobiology Program of The Department of Pathology, University of Maryland School of Medicine. LITERATURE CITED Barrett, L. A,, E. M. McDowell, A. L. Frank, C. C. Harris and B. F. Trump 1976 Long-term organ culture of human bronchial epithelium. Cancer Res., 36: 1003-1010. Bulger, R., and B. F. Trump 1968 Renal morphology of the English sole ( P n r o p h e y s u e t z i h s ) . Am. J. Anat., 123: 195-225. 1969a Ultrastructure of granulated arteriolar cells (juxtaglomerular cells) in kidney of a fresh and a salt water teleost. Am. J . Anat., 124: 77-87. 1969b Ca++and K+ ion effects on ultrastructure of isolated flounder kidney tubules. J . Ultrastruct. Res., 28: 301319. 1969c A mechanism for rapid transport of colloidal particles by flounder renal epithelium. J. Morphol., 127: 205-223. Burg, M. B., and P. F. Weller 1969 Iodopyracet transport by isolated perfused flounder proximal renal tubules. Am. J. Physiol., 21 7: 1053-1056. Forster, R. P. 1948 Use of thin kidney slices and isolated renal tubules for direct study of cellular transport kinetics. Science, 188: 65-67. 1953 A comparative study of renal function in marine teleosts. J. Cell Comp. Physiol., 42: 487-510. 1961 Kidney cells. In: The Cell. J. Brachet and A. E. Mirsky, eds. Academic Press, New York, Vol. 5, pp. 89-16], 1967 Renal transport mechanisms. Fed. ROC.,26: 1008-1019. Forster, R. P., and F. Berglund 1956 Osmotic diuresis and its effect on total electrolyte distribution in plasma and urine of the aglomerular teleost, Lophizrs nmericnniis. J. Gen. Physiol., 39; 349-359. 1957 Contrasting inhibitory effects of probenecid on the renal tubular excretion of PAH and on the active reabsorption of urea in the dogfish, Sqiinlus ncnnthins. J. Cell. Comp. Physiol., 49: 281-285. Forster, R. P., and S. K. Hong 1958 I ~ znitro transport of dyes by isolated renal tubules of the flounder as disclosed by direct visualization. Intercellular accumulation and transcellular movement. J. Cell. Comp. Physiol., 51: 259-272. Forster, R. P., I. Sperber and J. V. Taggart 1954 Transport of phenolsulfonphthalein dyes in isolated tubules of the flounder and i n kidney slices of the dogfish. Comparative phenomena. J. Cell. Comp. Physiol., 44: 315-318. Hickman, C. P., and B. F. Trump 1969 The kidney. In: Fish Physiology. W. S. Hoar and D. J. Randall, eds. Academic Press, New York, Vol 1, pp. 91-239. Enter, W. B. 1966 Chlorophenol red influx and
eflux: microspectrometry of flounder kidney tubules. Am. J. Physiol., 211: 1152-1164. Puck, T. T., K. Wasserman and A. P. Fishman 1952 Some effects of inorganic ions on the active transport of phenol red by isolated kidney tubules of the flounder. J. Cell. a m p . Physiol., 40: 73-88. Sahaphong, S., and B. F. Trump 1971 Studies of cellular injury i n isolated kidney tubules of the flounder. V. Effects of inhibiting sulfhydryl groups of plasma membrane with the organic mercurials PCMB (parachloromercuribenzoate) and PCMBS (parachloromercuribenzenesulfonate). Am. J. Pathol., 63: 277-298. Shelburne, 3. D., A. U . Arstila and B. F. Trump 1973 Studies on cellular autophagocytosis. The relationship of autophagocytosis to protein synthesis and to energy metabolism in rat liver and flounder kidney tubules in uitro. Am. J. Pathol., 73: 641-4570. Trump, B. F., and A. U. Arstila 1975 Cellular reaction to injury. In: Principles of Pathobiology. M. F. LaVia and R. B. Hill, eds. Oxford University Press, New York, Second edition, pp. 9-96. Trump, B. F., and R. E. Bulger 1967 Studies of cellular injury in isolated flounder tubules. I. Correlation between morphology and function of control tubules and observations of autophagocytosis and mechanical cell damage. Lab. Invest., 16: 453-482. 1968a Studies of cellular injury in isolated flounder tubules. 111. Light microscopic and functional changes due to cyanide. Lab. Invest., 18: 721-730. 1968b Studies of cellular injury i n isolated flounder tubules. IV. Electron microscopic observations of changes during the phase of altered homeostasis in tubules treated with cyanide. Lab. Invest., 18: 731-739. 1971 Experimental modification of lateral and basilar plasma membranes and extracellular compartments in the flounder nephron. Fed. Proc., 30: 22-41. Trump, B. F., and F. L. Ginn 1968 Studies of cellular injury i n isolated flounder tubules. 11. Cellular swelling in high potassium media. Lab. Invest., 18: 341-351. 1969 The pathogenesis of subcellular reaction to lethal injury. In: Methods and Achievements in Experimental Pathology. E. Bajusz and G. Jasmin, eds. Yearbook Chicago, VO~. 4, pp. 1-29. Trump, B. F., R. T. Jones and S. Sahaphong 1975a Cellular effects of mercury on fish kidney tubules. In: The Pathology of Fishes. W. E. Ribelin and G. Migaki, eds. University of Wisconsin Press, Madison, pp. 585-612. Trump, B. F., J. M. Valigorsky, R. T. Jones, W. J. Mergner, J. H. Garcia and R. A. Cowley 1975b The application of electron microscopy and cellular biochemistry to the autopsy: Observations on cellular changes in human shock. Human Pathol., 6: 499-516. Trump, B. F., W. J. Mergner, M. W. Kahng and A. J. Saladino 1976 Studies on the subcellular pathophysiology of ischemia. Circulation, 53: Suppl. I, 17-26. Wasserman, K., E. L. Becker and A. P. Fishman 1953 Transport of phenol red in the flounder renal tubule. J. Cell. Comp. Physiol., 42: 385393.
