Hypertrophy of renal mitochondria

822 Volume I ‘Number 5’ 1990

Hypertrophy of Renal Mitochondria

Soon Hwang, Roger Bohman, Pat Novas, Jill T. Norman, Timothy Bradley, and Leon G. Fine1

S. Hwang. P. Navas. Department of Biology, Universityof California, Los Angeles

R. Bohman, J.T. Norman, L.G. Fine, Division of Nephrology,Department of Medicine, University of California, LosAngeles

T. Bradley. Department of Ecology and Evolutionary

Biology, University of California, Irvine

(J. Am. Soc. Nephrol. 1990; 1:822-827

ABSTRACTCompensatory renal hyperfrophy leads to an in-crease in the size and metabolic capacity of renaltubular cells. Increased transport and metabolic ac-

tivities must be sustained by an augmented rate of

energy production, which is largely dependent onmitochondrial processes. Although previous studies

have suggested that mitochondria proliferate in thehypertrophying cell, the data to support this have

not been convincing. This study was designed to

determine whether the mitochondria of the hypertro-phied renal proximal tubular cell undergo hypertro-phy or proliferation. Flow cytometric analysis of prox-

imal tubular cells obtained from the kidneys of

uninephrectomized rabbits revealed an increase incell size and RNA content compared with controlcells but showed no change in DNA content andnuclear size and no evidence of entry into the S/G2/

M phases ofthe cell cycle. Histomorphometric analy-

sis of cortical proximal tubules revealed that al-

though cytoplasmic volume increased, mitochon-

drial density remained constant, indicating that mi-

tochondrial volume increases in proportion to the

increase in cell volume. By day 14, mitochondrialvolume had increased 66% above control values.Electron microscopic examination of isolated S2 prox-

imal tubules from 5/6 nephrectomized rabbits with

maximal hypertrophy revealed mitochondrial cristaewhich appeared to be more densely packed thanthat in normal cells. The size of the functional mito-

chondrial pool per cell was determined by rhoda-

‘Correspondence to Dr. L.G. Fine, Division of Nephrology, Department of Med-icIne, University of California, Los Angeles, School of Medicine, Los Angeles. CA90024.

1046-6673/01 05-0822$02.00/0Journal of the American Society of NephrologyCopyright © 1990 by the American Society of Nephrology

mine-123 fluorescence. This increased within 24 h ofuninephrectomy, peaked at approximately 80%above control levels at 5 days, and remained ele-vated throughout the 16 days of observation. Theinitial increase (days I and 2) occurred before ameasurable increase in mitochondrial volume oc-curred and presumably reflects an increase in mito-chondrial membrane potential. Southern analysis ofDNA hybridized with a single-copy nuclear gene(apolipoprotein B) and with the canine mitochon-drial genome showed a constant hybridization signal

for both probes from I to 32 days after uninephrec-

tomy, indicating that the copy number of thesegenes does not change. On the basis of the in-creased rhodamine-123 fluorescence per cell, theincreased mitochondrial volume by histomorphom-etry, and the absence of evidence of multiplicationof the mitochondrial genome, we conclude that themitochondria of the hypertrophied proximal tubular

cell undergo true hypertrophy and do not proliferateafter unilateral nephrectomy.

Key Words; Rhodamine- 123, proliferation, histomorphometry,

flow cytometry. unilateral nephrectomy

W hen the renal tubular cell undergoes compen-satony hypertnophy after reduction of the

nephnon population, a state of “hypenmetabolism” isinduced ( 1 ). Oxygen consumption per nephron is in-

creased, linked both to increased transcellular so-dium transport and to other cellular processes.

Histomonphometnic analysis of the remnant kidney

has shown that mitochondnial volume increases in

proportion to the overall increase in cell volume, i.e.,

mitochondnial “density” remains constant (2). Theyield of mitochondnial protein from the remaining

kidney increases within the first 3 days after unilat-

enal nephnectomy as does the apparent rate of mito-chondnial protein synthesis (3).

On the basis of studies which have measured ml-

tochondnial profiles, i.e. , mitochondnial count per tu-bule section (4), mitochondrial DNA synthesis (5), and

non-nuclear DNA (6), it has been proposed that ml-tochondnial proliferation occurs in compensatoryrenal hypertnophy. This implies that mitochondriaundergo replication. If this were true, it would provide

a partial explanation for the finding that total kidneyDNA content increases marginally after renal abla-

tion in adult animals (7). On the other hand, while

Hwang et al

Journal of the American Society of Nephrology 823

the conclusion that mitochondnia replicate them-

selves is convenient, its implication is far ranging,

implying that independent control of nuclear and

mitochondnlal genomes may exist In the hypentro-

phied cell.To address the question of whether the increase in

mltochondnial function, size, and apparent numberIs due to hypertrophy or to proliferation (replication),we quantitated mitochondriab function and gene copy

number during the course of compensatory renalhypertnophy. Our findings indicate that renal tubularmitochondnla do not undergo replication but ratherundergo hypertnophy as part of the compensatory

growth response. This is the first instance, to our

knowledge, In which true hypertnophy of mitochon-dna has been documented.

MATERIALS AND METHODS

Animals

Male New Zealand White rabbits weighing approx-imately 1 .8 kg were subjected to left unilateral ne-

phnectomy as previously described (8). The remainingkidney was removed for study after 1 , 2, 4, 8, 16,

and 32 days. The left kidneys served as controls.

Isolation of Proximal Tubular cells

A suspension of single proximal tubular cells wasisolated by a modification of a previously described

method (9). In brief, the renal artery was cannulatedand the kidney was perfused with Hanks’ solution toremove blood. The kidney was then infused with a

solution containing iron oxIde, excised, and decap-sulated. CortIcal tissue was cut into pieces, washed

twice with PBS and suspended in Hanks’ solutioncontaining 0. 1 % type II coblagenase (Sigma ChemicalCo. , St. Louis, MO). This mixture was incubated at37#{176}Cfor 15 mm In a shaken bath. After the incuba-

tion, tissues were mechanically disaggnegated, fib-tered through 80-sm-pore-size mesh, and the glomen-uli, containing trapped iron oxide particles, were ne-moved with a magnet, leaving a proximal tubulesuspension. This was centrifuged at 500 x g for 2mm, the supennatant was discarded, and the pelletresuspended in 50 ml of a hypotonic buffer (75 mos-

mol/kg) devoid of divalent cations which contained(In mM): KC1, 5; Na2PO4, 1 ; D-glucose, 5; L-lactate, 1;

L-alanlne, 1 ; NaHCO, 26; and BSA, 0.2% (wt/vol) (pH7.35 to 7.40). The suspension was mechanically ag-

Itated In a shaken bath for 60 to 90 s to release singlecells. These were then pelleted and washed as de-

scnibed previously.Cell numbers were determined on the final pnoxi-

mal tubular cell suspension by using a Coulten coun-

ter (Coubten Electronics, Inc., Hlaleah, FL). On thebasis of these counts, a gIven number of cells wasfixed In 70% ethanol for nucleic acid staining.

Staining of Cells with Fluorescent Probes

Cells fixed in 70% ethanol at 4#{176}Cfor staining ofnucleic acids were divided into samples of 2.0 x 106

cells. Samples were washed twice with cold PBS.RNAase A (Sigma) (0. 1 mL) (0. 1 mg/mL in PBS) was

added to half of the sample; to the other half, 0. 1 mLof PBS was added. For measurement of DNA andRNA content, cells were incubated for 30 mm at 37#{176}CIn 0.9 mL of propidium iodide (Sigma) solution (50

,zg/mL) in hypotonic sodium citrate. For measure-ment of mitochondniab activity, separate samples of2.0 x 106 nonflxed cells were incubated for 30 mm

at 37#{176}Cin PBS containing 1 .0 �g of rhodamine- 123

per mL (Eastman Kodak Co., Rochester, NY) (10). Thecells were examined by epifluonescence microscopyto determine the distribution of rhodamine- 1 23 flu-onescence within the cell.

After analysis of the propidium iodide-stained cellsby flow cytometry, samples were treated with 0. 1 mL

of ZAP-Isoton (Coulten Electronics) at room tempera-tune for 20 mm. ZAP-Isoton byses plasma membranesleaving nuclei intact. The isolated nuclei were rean-

alyzed by flow cytometry. RNA content per cell wasdetermined as the difference in pnopidlum Iodide flu-

onescence between untreated cells and cells treatedwith RNAase.

Flow Cytometric Analysis

Cells stained with dyes were analyzed on an EPICSV cell sorter (Coulten Electronics) coupled to an argon

laser (Coherent Lasers, Palo Alto, CA). A minimum

of 1 0,000 cells were analyzed in each sample. Theexcitation wavelength was always 488 X. For samples

stained with nhodamine- 1 23, fluorescence emissionwas monitored at wavelengths above 530 A. To allow

for day-by-day comparisons of stained samples, the

instrument sensitivity was adjusted to a constant

distribution by using standard fluorescent micro-spheres. Rhodamine- 1 23 fluorescence was then ana-lyzed at the same settings. Mean values of distnibu-tions were obtained by using the statistical packageof the MDADS Computer (Coulten Electronics).

Cell cycle distributions were obtained by collectingpropidium iodide fluorescent emissions above 590 A

as described previously (8). Cell doublets were elec-tronicably excluded by gating on the pulse versusintegral fluorescence distribution. The percentage ofcells in each phase of the cell cycle was determinedby using programs in the MDADS computer (Coulten

Electronics).

DNA Restriction, Gel Electrophoresis, andHybridization

Genomic DNA was prepared by the method de-scnibed by Maniatis et al. ( 1 1 ) and was digested with

Penal Mitochondrial Hypertrophy

824 Volume I . Number 5. 1990

either EcoRI or HindIII (Bethesda Research Labona-tories, Inc. , Gaithersbung, MD) and separated on 0.8%agarose gel. DNA was vacuum blotted onto Z-probefilters (Blo-Rad Laboratories, Richmond, CA). Prehy-bnidization, DNA-DNA hybridization, and washeswere performed as described by Blo-Rad. Autoradi-ography was carried out at -70#{176}C by using KodakXAR film with Intensifying screens, and DNA hy-

bnidization intensities were measured by using a Blo-Rad video densitometer (model 620) combined with

IBM software.

DNA Probes

The human apolipoprotemn B (apo B) gene was usedas a probe for the nuclear genome (1 2) (provided byDr. J. Lusis, University of California, Los Angeles).The human apo B probe consists of a 3.4-kb EcoRIcDNA fragment in pBR322. The canine mitochon-

driab genome (total of 1 7 kb) was used as the mito-chondnial probe (provided by Dr. R. Wayne, Univer-

sity of California, Los Angeles). Two of the threeBamHI fragments are cloned in lambda Embl3 (7.5

and 8.5 kb, respectively). The remaining 1 kb iscloned in pUC19. The EcoRI apo B Insert and theInserts comprising the entire cloned mitochondnial

genome were 32P-labebed with a random-primed DNA

labeling kit from Boehringer Mannheim Blochemi-cals (Indianapolis, IN). Both the nuclear and the ml-tochondnial gene probes were used on the same DNAsample obtained at each time point.

Histomorphometry and MitochondrialMorphology

The renal arteries of control and contralaterab kid-

neys were cannulated, and the kidneys were perfu-sion fixed for 10 mm with cold 2.5% glutaraldehyde

(Sigma) in 0. 1 M sodium cacodylate buffer (pH 7.4)containing 5% sucrose. Sections of superficial cortexwere fixed for 2 to 4 h at 4#{176}Cand were then washed

and stored in sodium cacodylate buffer. Tissues werepostfixed In 1 % osmium tetroxide in 0. 1 M sodiumcacodybate buffer and processed for electron micros-copy as described previously (2). Sections ( 1 00 nm)were stained with saturated uranyl acetate and lead

citrate, coated with carbon, photographed (magnifi-

cation, X40,000), and analyzed histomorphometri-cally as described previously (2). Tubular cytoplasmicarea was measured on cross sections of tubules with

toluldine blue-stained sections photographed at amagnification of xl ,400. Tubule volume is expressed

as volume of cytoplasm pen 1 zm of tubule length.Mitochondniab density indicates the percentage of cel-lular volume occupied by mitochondria, whereas ml-

tochondnial volume represents the volume of mito-chondnla per micrometer of tubule length.

Mitochondnial morphology was studied on maxi-mally hypentrophied, remnant kIdneys of �/6 nephrec-

tomized rabbits with material derived from a previous

publication (2). In these studies, fixation for electronmicroscopy was performed on isolated proximal tu-bule segments removed 1 8 to 25 days after nephrec-tomy and perfused in vitro to maintain active trans-tubular transport. The method of perfusion and fix-

ation for histomonphometnic characterization ofthese tubules is described in a previous publication

(2) which did not include morphological characteriza-tion of the mitochondnia.

RESULTS

Flow Cytometric Analysis

Hypertnophy of renal proximal tubular cells wasdemonstrated by flow cytometry with the kidney re-moved at zero time (i.e. , the uninephrectomy) as con-trob. The time course of the change in cell size isshown in Figure la. This Increased to a maximum ofapproximately 80% above control by day 5 and ne-mained constant thereafter for the duration of the

experiment. Nuclear size, In contrast, was un-

changed oven the 1 6-day period of observation.Mitochondrial content, per cell, was quantitated by

rhodamine- 1 23 fluorescence (Figure 1 b). Rhoda-

mine- 1 23 fluorescence was increased by day 1 at atime when cell size was not different from control. Itincreased further to a maximum of approximately

80% above control at 5 days and stabilized at roughly50% above control for up to 1 6 days postnephnec-

tomy.Cellular RNA content (Figure ic) paralleled the

changes in cell size, whereas DNA content (Figureid), reflected the position of the cells In the cell cycle

as being unchanged, with cells remaining in G0/G1

throughout the 16 days of observation.

Histomorphometry and MitochondrialMorphology

Histomorphometnic analysis (Table 1 ) showed thatmean mitochondnial volume pen micrometer of tubulelength increased from 230 ± 46 to 383 ± 48 �m3 by

day 14. Despite the fact that rhodamine fluorescenceincreased within the first 48 h after unlnephnectomy,

there was no measurable increase In mitochondnialvolume during this period. The morphology of themitochondnia of S2 proximal convoluted tubules from

�/6 nephnectomlzed rabbits is shown in Figure 2. Themitochondria of the remnant kidney appeared nor-

mal In all respects and showed no evidence of swell-Ing. The cnistae of mitochondnia from remnant kid-neys appeared to be more densely packed than those

of normal mitochondnia, but this was not quanti-tated. The mitochondnial volume of these tubules has

Numberof cells

Cell 9O�,/1ie�II� Usize

30

b120

Rhod-123 80

fluorescence

40‘ I

C

:��,///?“���‘s,%,,whoIe cellsRNA ______________content

�-�c��IeI

15

% cells 10S/62/M

a Mean ± SE of 8 to 10 measurements.

b p < .0 1 versus control

Hwang et al

Journal of the American Society of Nephrology 825

0 5 10 15

Intensity Days postnephrectomy

Figure 1. Cell and nuclear size, rhodamine-123 fluores-cence, cellular and nuclear RNA content, and cell cycleanalysis of renal proximal tubular cells. The left panel showsthe raw data plotted as fluorescence intensity versus thenumber of cells obtained from control cells before unine-phrectomy. The right panel shows the time course for thederived data from I to 16 days postuninephrectomy: (a)Cell and nuclear size measured by forward-angle light scat-ter; (b) rhodamine-123 fluorescence in intact cells; (c) pro-pidium iodide fluorescence of intact cells and isolatednuclei with and without RNAase treatment (left panel). Thedifference between the two values is plotted on the rightfor whole cells and for isolated nuclei; (d) frequency distri-bution of propidium iodide fluorescence in intact cells. Thequantity of DNA per cell (proportional to fluorescence in-tensity) is shown for the different phases of the cell cycle(G0/G1, 5, G2/M) in the left panel. Cells containing DNA inexcess of the G0/G1 amount are considered to be ‘cycling”cells. The panel on the right represents the percent ofcycling cells in the G2/S/M phases of the cell cycle.

been reported previously to increase by a mean of

82% above control levels (2).

Southern Analysis

Densitometric scanning of autonadiognaphs ofSouthern blots revealed that the apo B gene signalremained constant throughout the 32 days of obser-

vation and was the same as that in control kIdneys(Figure 3). Six different mltochondnlal bands were

quantitated as shown in Figure 3; these too were

unchanged throughout the 32 days of observation.

Since a fixed amount of total DNA was loaded perlane and cellular DNA content remained constant(Figure 1 d), it is evident that the ratio of mitochon-

TABLE I . Histomorphometric analysis of mitochondrialdensity in superficial proximal tubulesa

TubuleCytoplasmic

Volume(Mm3)

MitochondrialDensity

%

MeanMitochondrialVolume (Mm3)

Control 790±65 29.2±1.1 230±4624h 933±204 25.4±3.0 232±30

48 h 701 ± 79 27.3 ± 0.8 189± 21

14 d 1,317 ± 133b 29.1 ± 2.3 383 ± 48b

Figure 2. (a) Electron micrograph ofthe basal region of thecell from the 52 proximal covoluted tubule of a normalrabbit kidney. The mitochondria associated with the basalinfolds have well-developed cristae (arrowhead) which arerelatively widely spaced within the mitochondrion. Barequals 0.5 micrometers. (b) Electron micrograph of thebasal region of the cell from the convoluted tubule of aremnant kidney. The mitochondria associated with thebasal infolds have matrices which are more condensedthan those from normal preparations. As a result, the mem-branes of the cristae are difficult to discern but the spaceswithin the cristae are very evident (arrowhead). The cristaein these mitochondria appear to be more densely packedwithin the mitochondrion than those in the tubules of normalrabbits. Bar equals 0.5 micrometers.

dnial to nuclear gene copy number was constantthroughout the course of the experiment.

DISCUSSION

The increases in mitochondnial volumes and pro-

files which have been observed In compensatory hy-pertrophy of renal tubular cells have been ascribedto proliferation (replication) of mitochondnia (3-6).

Renal Mitochondrial Hypertrophy

826 Volume I ‘NumberS#{149} 1990

APO- B

MITOCHONDRIAL

0 I 2 4 8 632

DAYSFigure 3. Southern analysis of renal cortical DNA. Upperpanel. apo B, autoradiography of nuclear DNA hybridiza-tion. Cortical DNA was digested with EcoRl and 50 �g wasloaded per lane. DNA was electrophoresed on a 0.8%agarose gel for 220 mm at 100 V. Hybridization with (32P)-labeled human apo B cDNA was performed for 48 h at 55#{176}C.Lower panel. Mitochondrial, autoradiography of mitochon-drial DNA hybridization. Cortical DNA (the same samples asthose used for the nuclear DNA hybridization shown above)was loaded at 5 �g per lane and electrophoresed as de-scribed above. Hybridization with (32P)-labeled mitochon-drial plasmid was performed for 24 h at 55#{176}C.The sizes ofthe restriction fragments in both panels are shown on theright in kilobases.

This conclusion was made on the basis of an in-

creased yield of mitochondnial protein, an increaseIn I3Hlleucine incorporation into mitochondnial pro-tein, and an increase in nonmitochondnial DNA. The

demonstration by Ch’ih and Devlin (5) that there isan apparent increase in renal mitochondnial DNA

content at 24 h with a return to baseline at 36 h

would suggest that at least one cycle of mitochondnial

replication must have occurred at an early point intime. However, since there was no regression of the

hypertrophy after 24 h, this latter finding is difficult

3 8 to understand, since mitochondnial volume is in-‘ creased as long as the kidney remains hypertnophied

(2).In this study, histomorphometnic analysis revealed

that an increase in mitochondnial volume occursafter unmnephrectomy. This approach, however, can-

not differentiate between enlargement/elongation

versus proliferation of mitochondnia-both of which

would manifest as an increase in mitochondnial vol-ume per unit length of tubule. Given that mitochon-

6. 9 dna may employ the method of genome amplificationto increase rRNA production rather than maintainingmultiple copies of nibosomal genes pen genome (13)

and given a previous report describing an increase in3 . 4 non-nuclear DNA in this model, the possibility that

mitochondnia replicate during hypertnophy seemed

2 . 6 feasible. We used the approach of estimating genecopy number to determine whether on not replication

of the mitochondnial genome occurs during compen-

I 4 satony hypertrophy by using a single-copy nuclear

I . gene as a normalizing control. Our results are con-

sistent with our earlier findings in this model, which

I . 0 indicated that there is no entry of cells into the cellcycle and no increase in total cellular DNA content

O 7 (8). Southern analysis oven a 32-day period showed a

S constant signal for the single-copy nuclear gene, apo

B and for the mitochondnial sequences.This finding does not support the contention of

Cuppage et al. (6) that “during the cellular hypertno-phy, mitochondnia first proliferate in number and

then increase in size”. The reason for the differencein conclusion between their study and ours is pne-sumably on the basis of methodological considera-

tions. Cuppage et al. (6) employed a method whichseparates nuclear DNA from non-nuclear DNA, the

latter being presumed to represent mitochondnial

DNA. They measured thymidine incorporation into

the two DNA pools to reflect proliferative activity butdid not indicate what the proportion of nuclear tonon-nuclear pool was. By using flow cytometny ofwhole cells and isolated nuclei, we have found levelsof non-nuclear DNA which are cleanly artifactual,i.e. , �20% of total cellular DNA, despite stringent

RNAase treatment to eliminate contamination withRNA (S. Hwang and R. Bohman, unpublished obsen-

vations). It is thus probable that current methods

cannot be used to separate nuclear and mitochon-

dnial DNA reliably.

As anticipated, there was evidence for increasedfunctional mitochondnial mass in the hypertrophied

renal tubular cells as defined by rhodamine- 1 23 flu-orescence. Rhodamine- 123 is mitochondnion-spe-

cific probe (10). The attraction of the cationic rho-damine molecule to the relatively high negative elec-

Hwang et al

Journal of the American Society of Nephrology 827

tnlcal potential across the mitochondnial membrane

is thought to be the basis for its mitochondnion-

specific staining in living cells. Rhodamine-123 flu-orescence increased within the first 24 h after uni-

nephrectomy and remained elevated thereafter. It isof Interest, and possibly of Importance with regardto the cascade of events leading to cellular hypertno-phy, that this increase in mitochondniab function

preceded the Increase In mitochondnial volume meas-uned histomorphometnically. Given that all constitu-

ents of the cell increase in hypertrophy, an earlyincrease In energy production would be a logical early

event in this process.

Mitochondnial morphology in remnant kidney prox-

imal tubules was not obviously distinguishable fromthat of normal tubules with the exception that the

cnlstae were more densely packed and the matrixappeared to be more condensed (Fig. 2). This reflects

an Increase In inner mitochondnial membrane areawhich is of Interest, considerIng that the mitochon-dna lie In close proximity to the basolatenal mem-

bnane, the area of which is also increased in thismodel (2).

To determine whether the increased mitochondnial

mass pen cell Is due to the presence of an increased

number of mitochondria or to an enlargement of afixed number of mitochondnia, we used Southern

analysis of DNA to determine whether the copy num-

ben of mitochondnial genes changes during the course

of hypertnophy. The results show that the mitochon-drlal genome remains remarkably constant through-

out the 32-day period of observation as does total

cellular DNA content. The constancy of the signalobtained by hybridization with the cDNA for a single-copy nuclear gene (apo B) showed that the yield of

cellular DNA was unaffected by the hypertnophicprocess.

In light of these findings, it must be concluded that

mitochondnlal DNA replication does not occur in com-

pensatony hypertrophy of the renal proximal tubular

cell and that the increase in mitochondnial profiles,

size, and function must be ascribed to a process ofhypentrophy of a fixed number of mitochondnia. It Is

of interest that while this occurs, nuclear size re-mains constant, suggesting that the process of orga-nellar hypertrophy may be confined to those organ-

elles which participate actively in the function of the

enlarged cell.

ACKNOWLEDGMENT

These studies were supported by grant ROl DK34049 from

the National Institutes of Health.

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