Document VGM5Zp2YDzp9GN5DzxNDnQy8j

ULTRASTRUCTURALTRANSFORMATION IN MITOCHONDRIA ISOLATED FROM KIDNEYS OF NORMAL AND LEAD-INTOXICATED RATS R. A. GOYER and R. KRALL 1`Vom the Department of Pathology and Genetics Curriculum, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27515 ABSTRACT Mitochondria isolated from kidneys of lead-intoxicated rats have been shown to have de creased oxidative and phosphorylative abilities. The purpose of this study was to determine whether these abnormal mitochondria would undergo ultrastructural transformation during controlled respiration in the absence of phosphate acceptor (State IV), as previously demon strated for normal liver mitochondria. It was first shown that normal rat kidney mito chondria transforms from a condensed ultrastructural conformation to an orthodox con formation after 5 min of State IV respiration with pyruvate-malate substrate. Reversal to a condensed conformation follows stimulation of respiration with adenosine diphosphate (ADP). A large portion of kidney mitochondria from iead-poisoned rats do not change from condensed to orthodox conformation during State IV respiration. Other mitochondria do transform to the orthodox form but they rapidly degenerate. State IV respiration decreases as these few orthodox mitochondria disintegrate. The conclusion is that those mitochondria that do not undergo change in ultrastructurc have impairment of electron transport, and that those that do become orthodox have increased membrane lability and undergo de generation. INTRODUCTION Mitochondria in proximal tubular lining cells in the kidneys of lead-intoxicated rats have been shown to be structurally and functionally ab normal (1). The basilar mitochondria of these cells are swollen and have marginal cristae and few matrical granules, and there is' an associated decreased reabsorption of amino acids. After isolation these mitochondria show a decreased respiratory control ratio and partial uncoupling of oxidative phosphorylation in the presence of pyruvate-malate substrate (2). Osmotic swelling experiments and electron microscopy of the mitochondria suggest that the mitochondrial membranes have increased lability. Phosphoryla- tive ability is only partially improved by treatment in vivo with EOTA1. Wc have concluded, there fore, that kidney mitochondria from lead-poisoned rats have a deficiency in oxidative and phos phorylative abilities, as well as a defect in mem brane integrity. The present study was undertaken to determine whether the abnormal mitochondria isolated from 1 Abbreviations used in this paper arc: EDTA, ethylenediaminctctraaccdc acid; ADP, adenosine diphosphate; ADPtO, adenosine diphosphate to oxygen ratio; TMPD, tetramothyl-p-phcnylcnediamine; JEM, trade name for microscope produced by Japan Electrical and Optical Co., Ltd. 393 i ) the kidneys of lead-poisoned rats would undergo centrifuged at 12,000 for 10 min. The crude mito .< ullrastruclural transfonnation during controlled chondrial pellet was resuspended iu 30 ml of 0.25 M respiration in the absence of phosphate acceptor sucrose at pi t 7.4 and centrifuged again at 12,000 g (Stale IV), as demonstrated for normal liver mito chondria by Hackcnbrock (3). Transformation from a condensed to an orthodox conformation occurring during State IV is reversible by A DP stimulation of respiration (State III). This ultra- for 10 min. The washed pellet was resuspended in 1.5 ml of 0.25 m sucrose and stored in an ice bath. An aliquot was removed for protein determination by a biuret method. Oxygen consumption was determined polarogxaphically (5), and rate of oxygen uptake, ADPtO structural change appears to be dependent on an ratio, and respiratory control ratio were calculated intact electron-transport mechanism and is from oxygen electrode tracings (6). The reaction mix believed to be related to the energy-conserving ture contained 2.2 mM pyruvate and malatc, 4.4 n<u function of mitochondria. It is thought not to be an osmotically induced ultrastructural change (4). Since we are unaware of previous studies of ultrastructural transformation in kidney mitochondria comparable to reported studies of this transforma tion in liver mitochondria, our studies of path ological mitochondria were dependent upon our phosphate, and 0.16 m sucrose. For stimulation of State III respiration, I pinole of ADP was added 2 min after addition of mitochondria. All reagents were made with ion-free water and adjusted to pH 7.4 with Tris base. 0.15 ml of mitochondrial stock solu-tion (containing approximately 3 mg of protein) was added to the reaction mixture to make a final volume of 3.0 ml. being able to demonstrate this phenomena in For ultx-astructural studies State IV respiration and normal renal mitochondria. State III respiration were measured as above. The rates of oxygen consumption for the mitochondria MATERIALS AND METHODS The oxidative and phosphoxylative studies shown in Table I were performed on mitochondria isolated fx-om which the electron micrographs were made ax'e shown in Figs. 1 and 5. Each tracing of oxygen uptake represents a separate aliquot of stock mitochondria. At the time indicated by the arrow at the end of each from the kidneys of four 200-g Spi-aguc-Dawlcy rats tracing, 0.12 ml of 25% glutaraidehyde was added fed a diet of pulverized laboratory chow that con tained 1% lead acetate. Respiratory and ultrastruc tural studies were repeated on mitochondria from to the reaction mixture. This treatment stopped respiration instantly. The mixture with fixative was allowed to remain in suspension in the cold (4<'C) the kidneys of three other lead-fed rats. An equal for a minimum of 1 hr. The reacuon mixture was number of similar rats fed the same chow without lead acetate served as controls. Rats wexe sacrificed by decapitation and the kidneys were quickly excised; then centrifuged at 35,000 g for 5 min to form a pellet of mitochondria. A ixarrow strip was then cut from the center of the the capsule was peeled free and the medullary zone pellet and further fixed with 1 % osmium tetx-oxide in was separated from the cortex. The cortical portion acetate-Veronal buffer at pH 7.4, dehydrated, em watt then homogenized in 0.40 m sucrose containing bedded in Epon, sectioned with a Porter-Blum ultra- 0.1 mu EDTA and centrifuged twice at 600 g for 10 microtome, and examined with a JEM T-7 electron min. The supernatant solutions were combined and xiixci'oscope. TABLE I Oxidative and Phosphorylaltve Abilities of Kidney Mitochondria from Control and LeadFed Rats with Pyritvaie-Mulate Substrate* Rate of oxygen uptake /tatoms O/min/pcr g protein Mitochondria No. J Stow IV State III ADiyO Respiratory control ratio Control Lead-Fed 8 43.2 1.9 130.3 13.9 2.7 0.2 3.3 0.5 7 46.5 dc 6.9 101.8 14.0 2.1 do 0.2 2.2 0.3 p <0.15 p < 0.05 p < 0.05 p < 0.05 * Reaction mixture as described in text (Methods), f Number of measurements. Stock mitochondria are from four control and three lead-fed rats. I 391 Tun JouitNAr. o f Ce l l 1!io i,o c y Vo l u me 41, 1969 j DUP050312363 RESULTS. Mitochondrial Respiration Table 1 compares the respiratory rates of kidney mitochondria isolated from normal and leadintoxicated rats. Oxygen uptake of mitochondria from the lead-fed rats in die presence of pyruvatemalate substrate (State IV) is slightly increased, whereas State III respiration during phosphoryla tion is decreased. Likewise, ADPtO ratios and respiratory control ratios of kidney mitochondria from the lead-intoxicated rats are less than the comparable values obtained with control mito chondria. Ultrasiructural Transformation The respiratory rates of mitochondria obtained from kidneys of a control rat arc shown in Fig. 1. The time points at which mitochondria were fixed for Figs. 2, 3, and 4 are indicated. The mitochondria shown in Fig. 2 respired in State IV with pyruvate-malate substrate for only 1 min. Nearly all of the mitochondria have a dense, contracted inner compartment and an expanded outer compartment. The conformation of these mitochondria resembles the condensed ultrastructural conformation demonstrated in liver mitochondria by Hackcnbrock (3). After respiring in State IV for 5 min, the mitochondria from the control animals changed in ultrastructure from a condensed to an orthodox conformation (Fig. 3), which resembles the ap pearance of kidney mitochondria usually observed in tissue sections. The inner compartment is expanded, the inner membrane forms thin cristae. and the outer compartment is very narrow. 90% of the mitochondria examined were of the con formation shown in Fig. 3. Most exceptions were damaged mitochondria, single examples of which appear in both Figs. 2 and 3. . Fig. 4 was obtained from mitochondria 30 sec after Slate III respiration was stimulated by the addition of ADP. These mitochondria were allowed to respire in State IV for 4 min before addition of ADP. Approximately 80% of kidney mitochondria assume an orthodox conformation after 4 min of State IV respiration. If the mito chondria are allowed to respire a full 5 min before the respiration is stimulated with ADP, little or no respiratory control is obtained and many of the mitochondria appear damaged. However, if ADP is added after only 4 min of State IV respiration, oxygen uptake is stimulated and reversal to a condensed ultrastructural conformation occurs (Fig. 4). Similar experiments were repeated with kidney mitochondria from lead-intoxicated rats. The respiratory rates and the time points at which mitochondria were fixed for electron microscopy are shown in Fig. 5. Initial State IV respiratory rates were similar to those observed for the control mitochondria, but after approximately 3 or 4 min the rate of oxygen uptake decreased. This change in State IV respiration was a consistent finding in experiments with mitochondria from lead-intoxicated rats. ADP only weakly stimulated respiration after 2 min of State IV respiration and produced no increase in oxygen uptake if added after 3 or more min of State IV respiration. After 1 min of State IV respiration, the majority of the mitochondria from the lead-poisoned rats Fig u k e 1 Oxygen consumption of control kitjney mitochondria. Mito chondria for Figs. 2 and 3 sere fixed after 1 min and 5 min of State IV respiration, respectively. Mitochon dria for Fig. 4 were fixed after 4 rain of State IV respiration, addition of ADP (1 /nnole/S.l rag protein), and ) '"in oi State HI respiration. The reaction mixture was as described in text (Methods) except that ADP was added only as indicated. l l. f I Go y ek a n i> Iv r .u .t . Ullrtixlmclvrul Traneformnlion in Kidney Mitochondria 395, J DUP050312364 Fig u k e 3 Mitochondria fixed after 1 rain of State IV respiration as shown in Fig. 1. Mitochondria haw a condensed conformation indicated by the increased density and contraction of tiie inner matrix. The den sity or degree of condensation varies in different mitochondria. The inner membranes appear to be ad hered to the outer membranes at multiple points. The expanded clear areas within the mitochondria are continuous with the outer compartment as indicated by arrows. A swollen damaged mitochondrion (s) is present. X 33,000. (Fig. 6) are in the condensed conformation, which resembles the conformation of die control mito chondria in Fig, 2. However, after only 3 min of State IV respiration, about half of the mito chondria have become orthodox in conformation; the remaining mitochondria remain condensed (Fig. 7). Some of the mitochondria that have undergone ultrastructurai transformation to an orthodox configuration appear swollen and damaged. Continuation of State IV respiration seems to worsen the ultrastructurai appearance of the mitochondria, resulting in a higher proportion with ruptured membranes (Fig. 8). Nevertheless, many more mitochondria remained in the con densed conformation than were observed in preparations of control mitochondria after 5 min of State IV respiration. Similar results were ob tained on mitochondria isolated from three differ ent lead-intoxicated rats. DISCUSSION The experiment demonstrates that mitochondria isolated from rat kidney cortex undergo an ultrastructural transformation from a condensed to an orthodox form during State IV respiration. This transformation resembles the change in ultra structure described by Hackenbrock for liver mitochondria under similar circumstances (3). The present experiment differs from Hackenbrock's study in only minor aspects. We initially fixed the mitochondria in the reaction mixture with glutaraldehyde before later fixation with osmium tetroxide. Pyruvate-malate rather than succinate, was used for substrate and the tem perature of the reaction mixture was 25C rather than 30C. A pyruvate-malate substrate has been consistently used in previous studies of the phos-phorylating defect of mitochondria of Icadintoxicated animals in this laboratory. Recent studies suggested that mitochondria from lead- 336 Th e Jo u r n a l o p Ce l l Bio l o g y Vo l u me 41, 19G9 DUP050312365 f Fig u r e 3 Mitochondria fixed after 5 min of State IV respiration as shown in Fig. 1. Tile mitochondria have undergone ultrastructurnl transformation from condensed to orthodox conformation. The inner compartment is finely granular and homogeneous. The eristae and outer compartment (c) are thin. The variation in size is charucteiistic of mitochondria isolated from kidney. X 33,000. Fiu u h e '1 Mitochondria fixed after State IV respiration lias continued for 4 min followed by A1IPstimulatcd respiration (State III) for 30 sec as shown in Fig. 1. State IV to State III transition is ac companied by transformation of ultrastructuro from orthodox to condensed conformation in nearly all of the mitochondria. X 32,000. 397 V f I DUP050312366 f ft i f ( ( ! S I t. Fic u h e 7 Mitochondria from kidneys of tv lead-mtoxieiitcd rat fixed after 3 min of State IV respiration (Fig. 5). About 50% of the mitochondria are transformed to orthodox conformation. These mitochondria differ from control mitochondria with orthodox conformation (Pig. 3) in that crislac arc sparse and irregu lar (c). Many of the mitochondria arc swollen (s). X 32,000. I'V.'tJiiK 3 Mitochondria from kidneys of a lead-intoxicated rat after a min of State IV respiration (Fig. 5). The decrease in oxygen consumption occurring after 3 min of State IV respiration is accom panied by swelling and rupture of membranes (arrows) of many of the mitochondria that hare undergone trnnsfonnatioii to the orthodox conformation. .Many mitochondria persist in the condensed conformation. X 32,000. i 5; DUP050312367 Flo V k b .) Oxygen consumption of miloc-liondihi from kidneys of leadintoxirnled mis. Mitochondria for Figs. 6, 7, and 8 wore obtained after 1, 8, ami 5 min of Slate )V respira tion, respectively. Tlie reaction mix ture was as described in text (Methods'). Fig u u b 6 Mitochondria from kidneys of lcnd-intoxicatcd ml fixed after 1 min of State IV respiration (Fig. 5). The majority of the mitochondria are in the condensed conformation and do not differ from control mitochondria after 1 min of State.IV respiration (Fig, 2). X 32,000. intoxicated rats have normal phosphorylative abilities in the presence of succinate. The morphologic appearance of condensed kidney mitochondria differs from that of liver mitochondria only in that the dense inner com partment appears adhered or fixed to the outer membrane at more points than were suggested for liver mitochondria. Also, the time required for State IV condensed to orthodox transformation to occur is less for kidney mitochondria, 5 min rather than 15 min. The ortliodox ultrastructural con formation of State IV kidney mitochondria was rapidly reversible to a condensed conformation during State III, as occurs in liver mitochondria. 390 Tu b Jo u k x .v u o f Cium I3io i.o o y - Yo i.u mb -11, I960 V.S'*.....; 1-7-.---.-,- DUP050312368 The significance of the ability of mitochondria to undergo ullrastructural transformation is not completely understood. Hackcnbrock has demon strated that this morphologic change may Ire prevented by agents which impair electron trans port, such as cyanide or atiliinycin (4). The latter compound may be bypassed with TMPO, which restores electron flow and ultrastructural transformation. It has been suggested that these ultrastructural changes are electron-transport energized mcchanochemical transformations, rather than ion-induced osmotic ultrastructural transformations energized by electron transport (4). Mitochondria uncoupled with small amounts of diriitrophenol are more rapidly transformed from condensed to orthodox forms during State IV and arc not reversed by stimulating respiration with A13P (State III). Other workers have shown that that mitochondria from experimentally-induced hepatomas and fetal liver have an orthodox con formation when isolated in State IV but will not assume a condensed conformation in State III. Also, respiration of these mitochondria is not stimulated by ADP, which, suggests an inability to perform phosphorylation (7). It has been demonstrated that transformation from orthodox to condensed, conformation may occur when phosphorylation is stimulated in situ (8). Addition of amino acids and amino acids plus glucose to "rings" of mouse jejunum results in stimulation of respiration and a condensed ap pearance of mitochondria in tissue sections, again demonstrating that condensed mitochondria! conformation is associated with a high rate of coupled phosphorylation. The present study shows that ultrastructural transformation of mitochondria from kidneys of lead-intoxicated rats differs from that of control mitochondria. A portion or the mitochondria from kidneys of lead-intoxicated rats docs not change from condensed to orthodox configuration during State IV respiration. Other mitochondria do transform to the orthodox form but they rapidly degenerate. State IV respiration decreases as the mitochondria of orthodox configuration degener ate. Interpretation of these changes can, of course, only be predicated on the incomplete understand ing of ultrastructural transformation available at the present time. However, it is suggested that those mitochondria that do not undergo con densed to orthodox transformation during State IV respiration have impairment of electron transport. Those mitochondria that do transform to orthodox conformation have some impairment of phosphorylativc ability manifested morpho logically by increased membrane lability and eventual degeneration. The research upon which this publication is based was performed pursuant to Contract No. PH 43-68-74 with the National Institutes of Health, United States Public Health Service, Department of 1 lealtb, Edu cation and Welfare, and supported by Grant No. AM 12061 from the National Institute of Arthritis and Metabolic Diseases. Received Jor publication 23 October 1968, and in revised form 26 December 1968. REFERENCES 1. Go y er , R. A. 1968. Lab. Invest. 19:71. 2. Go y er , R. A., A. Kr a i.l , and J. P. Kimb al l . 1968. Lab. Invest. 19:78. 3. Ha c k e n b r o c k , G. R. 1966. J. Cell Biol. 30:269. 4. Ha c k e n b r o c k , G. R. 1968. /. Cell Biol. 37:345. 5. Cl a r k , L. C., Jr ., R. Wo l f , D. Gr a n g er , and Z. Ta y l o r . 1953. J. Appl. Physiol. 6:183. 6. Ch a n c e, B., and G. R. Wil l ia ms . 1955. J. Biol. Chem. 217:383. 7. Min t z, H. A., D. H. Ya w n , B. Sa f e r , E. Br e s n ic k , A. G. Lie b e l t , Z. R. Bl a il o c k , E. R. Ra b in , and A. Sc h w a r t z . 1967. J. Cell Biol. 34:513. I. Jas per , D. K., and J. R. Br o n k . 1968. J. Cell Biol. 38:277. 400 Th e Jo u r n a l o f Ce l l Bio l o g y Vo l u me 41, If DU P050312369