Cell death during the postnatal morphogenesis of the normal rabbit kidney and in experimental renal polycystosis

J. Anat. (1978), 126, 2, pp. 303-318 303 With 19figures

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Cell death during the postnatal morphogenesis of the normal rabbit kidney and in experimental renal polycystosis

J. A. GARCiA-PORRERO, J. L. OJEDA AND J. M. HURLIt Fromthe Departmentof Anatomy, Faculty of Medicine of Santander,

Santander, Spain (Accepted4July 1977)

INTRODUCTION

In a previous work (Ojeda, Barbosa & Gomez-Bosque, 1972) we have studied themorphological aspects and the evolution of the renal lesions of theexperimental polycystosis model of Perey, Herdman & Good (1967). Our observations showed that this model presents threeevolutionary phases. Thetubular cysts, whicharethe predominant lesion ofthe second phase, suffer a remarkable reduction in number and sizeduring the thirdphase. This reshaping of the tubular cystscanbeexplained eitheronthe basis of selectivegrowthinhibitionandalteration of cellmultiplication rates,orofexcessive celldeath,orboth.

Cell death during normal morphogenesis is a fact. Its morphology, localization andpossible utilityhave beenextensivelyreviewedby Gluicksmann(1951),Saunders (1966), Saunders &Fallon (1966) andOjeda (1974). Cell death has beenstudied in theregression of themesonephros: this isaclearexample of phylogeneticdegenera- tion; however, there isalack ofinformation about the role of cell death during the postnatal morphogenesis of themetanephros.

The pathogenesis of renal polycystosis induced by corticoidsis stillincompletely understood.

Perey etal. (1967) havesuggested thathypokalaemiaisimportantinthe aetiology of cyst formation. Crocker & Vernier (1970) and Crocker (1973), working with fetal and embryonic kidneys, havereportedthat alowconcentration ofpotassium in the organculture medium occasionallyproduces cystic dilatation oftheureteral bud. The role ofpotassium in thedevelopment ofpolycystic kidneysis not entirely clear,however(Resnick,Brown &Vernier, 1973).

Percy (1975) has observed that thepyrimidine analogues 5-iododeoxyuridine and cytosine arabinoside produces microcystic changes similar to the lesions of the rabbits postnatally treated with corticosteroids. The pyrimidine analogues and the corticosteroids (Ruch, 1969) impairnormal cellulardivision. This suggests the possibility that the polycystic experimental kidney model of Perey et al. has, in additiontohypokalaemia, apathogenesisrelatedtoadirect pharmacologicalaction of corticosteroids. Courrier & Cologne (1951) and Jurand (1968) have observed that themalformationsinduced by corticosteroidsarerelated to abnormalnecrotic areas.

Abnormal cell death, which is very prominent in teratogenesis (for areview see Saxen & Rapola, 1969 and Menkes, Sandor & Ilies, 1970), could be the basic cellular lesion in thepathogenesisof the renalpolycystosisinducedbycorticosteroids.

In the presentworkwehave studied, by meansoflight

and-electron

the normal and abnormal cell death which takes place (i) during the postnatal morphogenesis of rabbit kidney, and (ii) in the experimental renal polycystosis model of Perey et al. Histological and ultrastructural lesions not directly related tocell death havenotbeen reported inthis work.

Our observations emphasize the role ofabnormal cell death in the evolution of cystic lesions.

MATERIALS AND METHODS

In our study we have used litters of newborn rabbits (Oryctolagus cuniculus, Spanish giant variety). Somemembers ofeach litterweregivenasingle intramuscular injectionofmethylprednisolone acetate (20mg/kg);the others wereinjected withan equalvolumeof salinesolution.Moredetailed information hasbeen given previously (Ojedaetal. 1972).

A total of 51 rabbits between 3 and48daysoldwas used. Theanimals werekilled by decapitation under ether anaesthesia, and the kidneys processed for light and electron microscopy.

Forlightmicroscopy, kidneys fragments, including bothcortex and medulla were fixed in Carnoy solution. Then they were dehydrated, embedded in eitherparaffin or Paraplast, and serially sectioned transversely at 7 ,m. Thesections were stained by the Feulgen method and with Harris' haematoxylin-eosin.

For electron microscopy the kidneys were fixed by perfusion through the aorta with 3% glutaraldehyde in 0-2M cacodylate buffer at pH 7-3. (Prior to its use, possible contaminants were eliminated from the glutaraldehyde solution by treat- ment with activated charcoal.) After perfusion fragments of the kidney were im- mersed in fresh coldfixativefor anadditional 2hours, then rinsedin 0-2 M cacody- late buffer and post-fixed in 1% osmium tetroxide for another 2 hours. Tissue blocks werestained with uranyl acetate, dehydrated in agraded series of acetones and propylene oxide, and embedded in Araldite. Semithin sectionswere cut witha LKBultratomeIII, stained with0-1 %toluidinebluein 1 % sodiumboratesolution, and mounted inadrop ofAraldite underacoverglass. These sectionswereused for generalorientation,aswellasforidentification ofpossible cell deathareas.Ultrathin sections of selected areas were then made, mounted on uncoated copper grids, stained with lead citrate (Reynolds, 1963) and examined with a Philips EM 201 electronmicroscope.

RESULTS

Celldeath in the normalkidney

Cell death can be observed occasionally during the postnatal development of the rabbit kidney. It can be classified on the basis of its localization andfrequency as follows.

Fig.1.Two intertubulardead cells in thekidneyofa15dayscontrol rabbit. One of these cells (c) shows an irregular nucleus with large clumps of heterochromatin, swollen perinuclear spaceandcysticmitochondria. The other cell showsapycnoticnucleus(arrow). Scale,1,um.

Fig. 2.Intertubular dead cell ofa12dayscontrolkidney.Notethe close association between thedegeneratedcell andcollagenfibrils(arrows).Scale,2,um.

Fig. 3. Intertubularphagocytecontainingonedead cell withsignsofdigestion.Notethe bundles ofmicrofibrilsnearthephagocyte (arrow).4dayscontrolkidney. Scale,2,um.

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Intertubular cell death

Dead cells can be observedamongthe tubular portions ofthe developing neph- rons. Specific localization of such cells (e.g. indeveloping capillaries) couldnotbe determined. Theycould be classifiedinto three groups:

(1) The first kind ofdeadcell is themost frequent. The nucleus isveryirregular, with markedclumpingof nuclearmaterial. Themostconspicuousfeature is the wide separation ofthe two nuclear membranes, producing vesicles (Fig. 1). These cells present many cellularprocesses, some of which are extremely long. Mitochondria are few in number andfrequently look cystic, with an electron-lucid matrix. They resemble fibroblasts, but they lack thelarge amount ofgranular endoplasmic reti- culum and theprominent Golgi complex characteristicofsuch cells. In some cases these dying cells are closely related to collagen fibrils (Fig. 2). No degenerative lesionscould be observed in the neighbouring tubules (Fig. 1).

Fig.5.Dead cells inside dilatedterminalampullaeofacollectingtubule. 3daystreatedanimal.

Araldite-embedded section(1lum)stained with toluidineblue.Scale,50,tm.

Fig.6. Freeorganellesand cellular debris in thecavityofadilatedterminalampulla. Swollen mitochondriaandgranularendoplasmicreticulumcanbedistinguished.5daystreatedanimal.

Scale,0-25,tm.

Fig.7.Cytoplasmiclesionsinthe wall ofatubular cyst inakidney4daysafterinjection.Note themitochondrial vacuolizationand the dilatation ofgranular endoplasmicreticulum.i, inter- tubular deadcell.Scale,2 ,um.

Cell death in the normal andpolycystickidney 307

20-2

(2) Thesecond type of intertubular dead cell death shows a pycnotic nucleus with concentrated chromatic material, and the cytoplasmic organelles cannot be dis- tinguished (Fig. 1). These cells are very small androunded. Sometimes they appear to be surrounded by arms of cytoplasm from an apparently viable intertubular cell.

(3) Thethird type of dead cell (Fig. 3) has been ingested by a phagocyte (a Type 2 phagocyte following the classification of Ballard & Holt, 1968).

The morphology of these three types of dead cells suggests that in fact they representsuccessive stages in onecontinuous necrotic process.

Intertubular cell death is more frequent in the younger stages ofdevelopment, and ithas not been observed after the twentiethpostnatal day.

Cell death in themetanephric blastema

The metanephric blastema at different stages of development, and especially at the point offusion of a renal vesicle witha collecting tube, was carefully observed for signs of cell death. Only two deadcells inanimals 3-5 days old were ever seen with the electron microscope, suggesting that cell deathinthenormalmetanephric blastema is avery rare event.

Celldeath in thepolycystic kidney

Themorphological changes inthekidneyof arabbit treated with corticoidfollow the stagesdescribedpreviously (Ojedaetal. 1972).Fromthe thirdday afterinjection until aboutthe fifteenthdaythekidneydemonstratesimmaturity. It shows an exten- sive nephrogenic zoneand a progressive dilatation of the ampullar portions ofthe collectingtubes (Fig. 4). Fromthe fifteenth to about the fortiethday, tubularcysts become large. After this the kidneys show two types of cysts. The more abundant andcharacteristicare the glomerularcysts. Tubular cysts, thoughpresent, arerare.

Atall stages of cyst evolution cell deathcanbeseenby opticand electronmicro- scopy, the morphology and topography of the dead cells changing with time.

The stageofimmaturity

Fromthethirdday after injection the ampullateblindendsofthecollecting tubes show progressive dilatation. Dead cells (Fig. 5) and cellular debris (Fig. 6) can be seenby light microscopy in the cavities ofabout 60% ofthe tubularcysts forming betweenthe fourth and fifthdays (Fig. 4). By the end ofthe period ofimmaturity (twelfth day) the number of cysts showing dead cells and cellular debris has de- creased to about 40%. The cells forming the walls of theampullae show lamellar bodies and aremarkable vacuolization ofthecytoplasm.Themitochondriaand the granular endoplasmic reticulum are swollen (Fig. 7). Some cells show signs of irreversible deterioration. The nucleus shows a condensation and margination of chromatin, and the perinuclear cistema is widened. These cells can be considered as dying.

Fig. 8. Phagocytesandonefreedegenerated cell (arrow)inthe nephrogenictissue ofa5days treated animal. The dead cells (dc) ingested by the phagocytes are in course ofdigestion.

Scale,4,um.

Fig.9.Epithelialcellcontainingonedeadcell in which nucleus(n)andcytoplasm (c)canbe distinguished. Note the close association of the phagocyte to the neighbouring cells, and the lack ofdegenerative signsinitscytoplasm.m,microtubules; d, digestive vacuole withcell fragments.9days treated animal.Scale,2jim.

Cell death in the normal and polycystic kidney 309

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The above mentioned changes takeplacemainly during the first days afterinjec- tion, and they have not been observed in equivalent structures in control animals.

Fromthe sixth daycell deathisessentially restricted to themetanephrogenic mesen- chyme.

It must be noted that the aggregation ofnephrogenic tissue seems to be greater in the treated animals than in the control rabbits. This is clear in the semithin sections. In this nephrogenic tissue the dead cells are grouped in small clusters of 3-5 cells. The electron micrographs show two kinds of dead cell. Thefirst is small and dense and free (Fig. 8). All the elements ofthese cells show increased electron density,thechromatinisclumpedinto large polarplaques,thecytoplasm isvery poor inorganelles, andtheyappear very closely packed. A remarkablefeature is a great reductionin surface area.

The second kind of dead cellhasbeeningested byanepithelialcell of the metane- phric blastema. These epithelial phagocytes are very numerous, and they can be divided into two successive stages, following the classification ofBallard &Holt (1968). In the early stage(Stage 1) thereare 1-2 dead cells in each phagocyte (Fig.

9), and the dead cells show few signs of digestion. They exhibit a degenerating nucleus, in which the chromatic and non-chromaticmaterial is fragmented,and a cytoplasmwithdilatedendoplasmic reticulumand debris. The nature of the contents of the other digestive vacuoles in the phagocytic cells is not known. In the later stage(Stage 2)thenumberofingesteddead cellsincreases to2-4 in eachphagocyte, and the dead cells show signs of digestion. The nucleus andcytoplasm cannot be clearly distinguished. Some degenerating mitochondria, dense bodies and mem- branous materialcanbe seen in thedigestive vacuoles.

Electron micrography reveals certain general characteristics of the phagocytes.

They possess numerous freeribosomesandmicrotubules (Fig.9);their endoplasmic reticulumis very scarce; an extensiveGolgicomplex with associatedcoated vesicles is found in the cytoplasm, and the ultrastructure is generally very well conserved.

Degenerating phagocytes like thosedescribed in various other tissuesin necrobiosis werenever seen(Dawd &Hinchliffe, 1971; Hurle, Lafarga &Ojeda, 1977).

Frequently thephagocytes have a dark cytoplasm whichcontrasts with the light cytoplasm ofadjacent cells (Fig. 8). They appear closely associated with the meta- nephric epithelial cells. Thedigestivevacuoles ofthesephagocytesareusuallylocated at the basal pole. Frequently phagocytes can be seen located in theparietal layer of Bowman's capsule ofthe developing renal corpuscle (Fig. 10). No degenerative lesions orphagocytescanbe seen in thevisceral layer.

Wehave observed intertubular dead cells similar inmorphological characteristics

Fig.10. Immaturerenalcorpusclewithadigestivevacuole(arrow)in onecell ofthe parietal layer of Bowman'scapsule. The visceral layer does not showdegenerative lesions. 5 days treated animal.Scale,5,tm.

Fig. 11. Freemitochondria between the basement membranes oftwotubules. 1,lysosome. 15 daystreatedanimal.Scale,2,tm.

Fig.12.Cytoplasmic vacuolation in the cells of the wall ofonetubular cyst. Notethatdamaged cells alternate with normalcells(arrows).p,phagocyte. Araldite-embeddedsection(1 ,um), stain- ed withtoluidine blue. 20daystreatedanimal.Scale,25,tm.

Fig. 13.Epithelialcell from thewall ofonetubular cyst. Note thewidened perinuclear cistema (pc) and the extremely distended endoplasmicreticulum. L, lumen of the cyst; b, basement membrane; ly,lysosome. 20daystreated animal.Scale, 1,tm.

Cell death in thenormal andpolycystic kidney

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and frequency to those described in the control animals. In some cases we have found free cytoplasmic organelles among the basement membranes of developing tubularcysts(Fig. 11).

In the period of immaturity of the polycystic kidney neither infiltration with hematogenous cellsnor signs ofinflammation were observed.

The stage oftubular cysts

At this stage the metanephrogenic zone has disappeared. Dying and dead cells can be seen in the walls of tubular cysts and among the basement membranes of adjacenttubules.

Within the wall of the tubular cysts, among healthy epithelial cells, groups of

Fig. 15.Lysosomesinthe wall ofatubular cyst inakidney 20 days after injection. Two swollen mitochondria(arrows)are tobeseen.Scale, 0-25 ,um.

Fig. 16.Myelin-like figureinalysosomeofthe wall of a tubular cyst. 22 days treated animal.

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Fig. 17. Twodigestivevacuoles(arrows) in the wall of a tubular cyst in a kidney 22 days after injection.Araldite-embedded section(1gim)stained with toluidine blue. Scale, 25,im.

Fig. 18.Freedeadcell in the lumen ofatubular cyst in a kidney 26 days after injection. Note the wellpreserved nucleus and the Golgi apparatus (arrow). Scale2,um.

Fig. 19.Cystproduced by dilatation of Bowman's capsule. Amorphous and cellular debris are observed in the lumen(L).37days treatedanimal. Scale, 5,im.

Celldeath in thenormal andpolycystic kidney 313

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10-15cellsshowing cytoplasmicalterationssuggestiveofdegenerationcanbe found;

these dying cells are located mainly in regions where two or more cysts comeclose together. Between the twentieth and the twenty sixthday after injection about 30% of the tubular cysts have degenerated cells in their walls. The most conspicuous featureofthedyingcells at thelight microscopic levelis thevesiculation of the cyto- plasm(Fig. 12). The electronmicroscope showsthat some of the largetubularcysts are lined by degenerating epithelium. The mitochondria of theseepithelialcellsare swollen and showareduced number ofcristae, beingfinallytransformed into empty vacuoleswithadecreased number of densegrainsin their matrix.Some cells showan extremely distended smooth-surfaced endoplasmic reticulum (Fig. 13). Free ribo- somescan also be observed. These lesions havenever been observed in the cells of the non-cystic collecting tube, whoseultrastructure is well preserved (Fig. 14), nor in the cells of thecollectingtubes of the control animals.

In the cytoplasm of degenerating cells we have found numerous dense bodies (Fig. 13) limited bya single membrane. Located between theelectron-dense matrix andthe membrane there isanelectron-lucid rim(Fig. 15). Someof thesedensebodies showmyelin-like figures, and thesecanbe observed incyst cells which do notshow any other type of lesion(Fig. 16).The nucleus shows moderate changes. Somecells show an indented nucleus with a swollen perinuclear space and vesiculation of the nuclearmembrane. Occasionally nuclei with densechromatin canbe observed. The changes mentioned above are frequently present in a single cell. Dead and dying cellsseem not tolose their attachmentstoadjacentcells.

Phagocytesarepresent in the walls of thetubular cysts(Fig. 17). These cellsareof epithelial origin and have ultrastructural characteristics similar to the ones ofthe period ofimmaturity described above.

Between the twenty second andthe thirty seventh day afterinjection about 12% of the tubular cysts are partially occupied by amorphous material, cellular debris and dead cells. Electron microscopy demonstrates a remarkable vacuolation of the cytoplasmof these cells.The mitochondria aswell as theendoplasmic reticulumare swollen and the cell membrane is broken; the structure of the nucleus remains unchanged, however (Fig. 18). Intertubular dead cells are present throughout the lifespan oftubularcysts.

Inthis period of tubular cysts, but more especially in the later stages, a second type of cystformedby dilatation ofthecapsularspace of Bowman canbe observed.

Thistypeof cyst reaches itsfull development inthe final stage in the evolution of the polycystickidney described by us (Ojedaetal. 1972). The walls of glomerular cysts are formed by an epithelium of flat cells typical of the parietal layer of the renal corpuscle. Inside the capsular cavity an amorphous material, and in some cases cellulardebris, canbeseen(Fig. 19).

DISCUSSION

Good fixation of kidney, and especially developing kidney (Larsson, 1975), is usually considered difficult, and so it might be tempting to some to interpret the cellular lesionsreported here as the consequences ofautolysis associated with poor fixation. Three arguments areagainstthis hypothesis. In the first place, degenerative lesions were seenin the kidney of control animals only in the interstitial cells and only during the first twenty days of postnatal development - never later. In the second place, we did not find degenerative lesions in the experimental animals, nor

Cell death in the normal andpolycystic kidney 315 inprenatal glomeruli whicharegenerallyconsidered difficulttofix (Johnston, Latta

&Osvaldo, 1973), nor in otherparts ofthekidney suchas the convoluted tubules, nor in the cells next to the degenerating ones. Finally, the images ofphagocytosis cannot be explainedas artefacts produced by apoorfixation. The observed lesions mustbeinterpreted asevidence of necrobiosis in the kidneys oflivinganimals.

Some of theintertubular dead cellsobservedin thekidneysofthe control animals can be equated to the 'cells with dark electron-dense nuclei' described by Leeson (1961) in theyounghamster. Bulger&Nagle(1973)havedescribed,in adult rabbits' kidneys, interstitial cells showing condensed chromatic material like the Type 2 cells described here. However, the cells described by us differ in such respects as scarcity orabsence ofGolgi cisternaeand lack oflipid droplets.

Macrophages have been observed in kidney interstitial tissue by several authors (Kirkman, 1943; Morrison &Schneeberger-Keeley, 1969;Romen &Thoenes, 1970);

however, phagocytes with ingested dead cells like those described in the present study havenotbeen reportedupto nowin the normalrabbitkidney.Leeson(1961), arguing from the decrease in the number of 'cells with electron-dense nuclei' with increasingage, suggested that such cells eitherdisappear completely or differentiate into other cell types. Buss &Gusek (1968) have observed that, during the postnatal development of the interstitial cells of rat kidney, initially undifferentiated cells are later transformed either into fibroblasts orinto cells presenting necrobiotic altera- tions similarto thosereported here. From our study it is possibleto postulate that deadcellseitherdisintegratein theintertubularspace or aredigested by phagocytos- ing cells.

Thepossiblerole ofintertubularcelldeathinthepostnataldevelopmentofkidney is not clear. It could indicate the disposal of a surplus ofundifferentiated meta- nephric cells thathave notparticipated inthedevelopmentofthekidney.Leeson(1961) suggested that basement membranes of the tubules derive partially from thisinter- tubularcell death; this is in agreement with our observations, for there wassome- times a clear association between the dead cells and the extracellular material, especially with the collagen fibres. Several authors (Hughes, 1943; Ojeda &Hurle, 1975) haveassociated cell death with theappearance of extracellularmaterial in the chick embryo, butexperimental proofis lacking.

Cell degeneration has been reported by Ilies (1971) during several stages of the development ofthe metanephros. In our study cell death was observed in thesub- corticalmetanephriczoneofnormal animals; its significanceis unknown.

The cell death observed in thekidney oftreated animals is abnormal inintensity and topography; nevertheless its role in thepathogenesis of this 'model' ofpoly- cystic kidneyis not clear. It is possible that cell death decreases too drastically the mass of cells in the metanephric zone; there are many examples (Grobstein, 1966, 1967)toindicate thatacertain minimummassofcells isoften necessary ifdifferentia- tion is to takeplace properly. Gossens & Unsworth (1972) havesuggested that, in mouse kidney tubulogenesis, the capacity for autonomous differentiation from the condensed stageto the elongatedoneand thento thecoiledtubule stage iscritically dependentuponthemassofavailablenon-induced mesenchymal tissue. Thedecrease of metanephric cell mass could explain the persistence of a nephrogenic zone and theprolongedrenalimmaturity, andperhapsthe formationof the glomerularcysts.

Furthermore, it hasbeenpointedoutbyus(Ojedaetal. 1972) that thepersistenceof the nephrogenic zone could explain the dilatation and excessive branching of the collecting tubules.

Apossible alternative explanation isthat cell death inthemetanephrogenic zone istheconsequence of poorinductionbythecystic collecting tube.

Cell death in the tubular cysts appears veryearly.Itispossible that thecelldetritus liberated into the cavity of the terminal ampullae retains water because of the oncotic pressure of its proteins, and this increases intracystic pressure. However, the cysts are always in communication with the tubular system (Ojeda et al. 1972) and, because ofthis,distensioncanhardly beadecisivefactor in thepathogenesisof this model ofpolycystickidney.

The cell death observed in the walls of the fully developed tubular cysts could, however, explain the change in the shape ofthe cysts that we have described pre- viously (Ojedaetal. 1972).

Themorphologicalchangesinthecystsbrought about bycelldeath couldbe very important, butitis verydifficultto deducethepathogenesisofthepolycystic kidney from themorphologyofthe lesions.

We can onlyspeculate about themechanism ofcell death inthis model ofpoly- cystic kidney induced by corticoids. Our electron microscope studies have shown that,duringtheperiodofimmaturity, thebasic lesionsarecondensation of chroma- tin and increase inthe number of free ribosomes. This type ofnecrosis is very fre- quently observed in teratogenic studies using substances which disturb replication, transcriptionandtranslation(Schweichel&Merker, 1973).

Corticosteroids can produce cell death in the kidney by their mitostatic effect (Ruch, 1969),and this isin agreementwith Kallen's theory(1965)thatcell death is a consequence ofaberrationsinmitosis.

Lamellarbodiessuch asthose observedinthe cells oftheterminal ampullaeof the treated animals have been attributed to impurities in commercial glutaraldehyde (Schultz &Case, 1970), butwith thisin mind weuseda highlypurified glutaralde- hyde. We have demonstrated a highcontent ofglycogeninthe walls of the tubular cysts (Garcia-Porrero & Ojeda, 1975), and on this basis the lamellar bodies could beattributedtothe transformationofglycogenintolipids (Curgy, 1968).

The pathological cell death in the kidneys of rabbits treated with methylpred- nisolone acetate is a paradoxical phenomenon, since corticosteroids have a cell- protecting effect attributed to their capacity to stabilize lysosomal membranes (Weissmann, 1975). However, insome cases itwould appearthatcorticosteroids are abletoprovokecellinjuryandevencelldeath.

At the stage of tubular cysts the dying cells show swollen mitochondria and a notable increaseinthenumber ofprimary lysosomes, supporting DeDuve's(1964) 'suicidebag'theory ofcell death.

Morrison & Panner(1964) observed thatexperimentalhypokalaemialeads to the formation of large numbers oflysosomes in the cells ofratrenal papillae. Wiener et al. (1968), using quantitative electron microscopy, found that cortisone induces a two tothreefold increase in the numberoflysosomes perunitarea ofcytoplasm, attributable to adecreased turnoveroftheseorganelles as aresult ofanincreasein their membrane stability. Thisfinding is in agreement withthe results obtained by Jurand(1968).

However, theprecise'causeand effect' relation betweenlysosomes and cell death is notclear.The release oflysosomalenzymesisprobablyamanifestationratherthan a cause of cell death (De Duve, 1964). It is likely that a combination of factors causescell death; e.g.changes in bloodsupplycanbeabasicmechanism.

Taken all in all the results ofthis study suggest that cell death is an important

Cell death in the normal and polycystic kidney

factor in the evolution of the lesions in the renal polycystosis induced by corti- costeroids, and probably in the genesis of this condition as well.

SUMMARY

We have studied, by means of optic and electron microscopy, the normal and abnormal cell death that takes place during the postnatal morphogenesis of rabbit kidney, and in the experimental renal polycystosis produced by methylprednisolone acetate.

In the normal kidney intertubular cell death can be observed during the first 20 days of the postnatal development. However, cell death in the normal metanephric blastema is a very rare event.

In the polycystic kidney numerous dead cells can be seen between the third and fortyeighth days afterinjection. The topography and morphology of the dead cells dependonthe stage in theevolution of the disease. In the 'stage of renalimmaturity', dyingand dead cells are present in the nephrogenic tissue, in the dilating collecting tubules and in the intertubular spaces. In this stage the cellular pathology is essen- tially nuclear. In the stage of tubular cysts, the dead cells are mostlylocated in the walls of cysts, with some dead cells, but mostly cellular debris in their lumina. At this stage the cellular pathology is basically cytoplasmic. The dead cells are even- tuallydigested by what appear tobephagocytes oftubular epithelial origin.

Itissuggested that celldeathis animportantfactor intheevolution of the lesions of renal polycystosis induced by corticosteroids, and probably in the initiation of thepathological process as well.

Support from the Marcelino Botin Foundation, Santander, Spain, is gratefully acknowledged.

The authors wish to thank Dr F. J. Garcia-Sancho for his invaluable help in the preparation of this manuscript.

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