Experimental Calcium Oxalate Nephrolithiasisin the Rat
Role of the Renal Papilla
SAEED R. KHAN, PhD,BIRDWELL FINLAYSON, MD, PhD,and RAYMOND L. HACKETT, MD
Calcium oxalate nephrolithiasis in rats, induced by asingle intraperitoneal injection of sodium oxalate, isassociated with pathologic changes in the renalpapillary tip. Calcium oxalate crystals appear in thetubular lumens, in the intercellular spaces betweenepithelial cells, and attached to the tubular epithelialbasal lamina. Unusual paracrystalline structures also
FOLLOWING a single intraperitoneal injection ofsodium oxalate (NaOx) calcium oxalate (CaOx)crystals can be observed in the rat kidney within 15minutes of injection."'2 Initially, crystals form onlythe lumens of proximal tubules but subsequently arefound in the papilla. Crystal size and retention areroughly dose-related.
Various hypotheses concerning calculus formationsuggest that the renal papilla has a central role; infact, the importance of the papilla is supported byboth morphologic and biochemical observations.Randall first emphasized the role of the papilla3'5when he described minute calculi and subepithelialcalcium plaques within and on human renal papillae,and suggested that these could serve as focal pointsfor stone growth. His results were substantiated byother investigators in a series of structural studies.6-8The critical importance of the papillary tip in the for-mation of various types of stones was further illus-trated in a number of experimental studies byVermeulen and co-workers.9'10 The demonstration ofa steep concentration gradient of calcium' 1-3 and ox-alate12 13 between the renal papilla, medulla, and cor-tex raises questions about the role played by thepapillary concentration gradient and its relation toboth the crystals formed proximally and the crystalmasses found in the papilla.
From the Department of Pathology and the Department of Surgery/Urology, College of Medicine, University of Florida, Gainesville, Florida
develop in the distal tubule associated with the basallamina. Speculations are made about the role of thesestructures. The epithelial changes are primarilynecrotic and are similar to those described in ex-perimental papillary necrosis. Complete morphologicrecovery occurs in 1-2 weeks. (Am J Pathol 1982,107:059-069)
To further investigate the role of the papilla incalculus formation, we examined the renal papillaeof rats with experimental CaOx nephrolithiasis, andin particular the papillary tip.
Materials and MethodsMale Sprague-Dawley rats weighing between 200
and 250 g, after at least 1 week of acclimatization toour animal quarters, were injected intraperitoneallywith 7 mg of NaOx per 100 g of body weight in a 0.22M NaOx solution in 0.9'70 saline. The kidneys wereexamined 1 hour, 3 hours, 6 hours, 24 hours, 48hours, 72 hours, 168 hours, and 336 hours after theNaOx injection. Normal rats in the same weightrange and housed under the same conditions wereused as control animals. All the animals were anes-thetized with intraperitoneal sodium pentobarbital.Kidneys were fixed by retrograde perfusion through
Supported by NIH Grant AM-20586-02.Accepted for publication November 24, 1981.Parts of this paper were presented as a Poster Session,
4th International Symposium on Urolithiasis Research,Williamsburg, Virginia, in June 1980.Address reprint requests to Raymond L. Hackett, MD,
Department of Pathology, College of Medicine, Universityof Florida, Gainesville, FL 32610.
the aorta"4 with a formaldehyde-glutaraldehyde mix-ture","6 for electron-microscopic examination, andby immersion in alcoholic formalin for light-micro-scopic examination.At least three rats were used for each time interval.
Light-microscopic (LM) examinations were performedon kidneys for all time periods. Scanning electronmicroscopic (SEM) examinations were performed on
kidneys 1 hour, 3 hours, 6 hours, 24 hours, 48 hours,72 hours, and 168 hours following NaOx injection;and transmission electron microscopic (TEM) exam-
inations were performed at 1 hour, 3 hours, 6 hours,and 72 hours following injection.
For LM examination, the upper and lower polesof the kidney were removed by sections passing throughthe pelvis above and below the papilla. The centralslice was embedded in paraffin, serially sectioned at 7 ,u,stained with hematoxylin and eosin, and examined bybrightfield and polarized light illumination on a LeitzDialux-20 microscope.For SEM examination, the papilla was removed
with fine scissors with care to avoid contact with thesurface. Selected papillae were examined and photo-graphed as whole mounts by light microscopy. Thespecimens for SEM examination were prepared ac-
cording to methods described earlier,2 sputter-coatedwith gold or gold-palladium, and examined on an
ETEC Omniscan or Hitachi 450-S SEM. First thepapillary surface was examined, and then the papillawas fractured longitudinally so that we could studythe interior. Kidneys to be examined by TEM were
processed as previously described."7 Because CaOxcrystals fracture during sectioning and tear the tissue,we dissolved the crystals in some specimens. Sampleswere taken from the primary fixative, rinsed severaltimes in buffer, and treated with 0.1 N HCI. Thetissues were examined under polarized light duringcrystal digestion; and when most of the birefringencedisappeared, the tissues were rinsed several times inbuffer, postfixed in osmium, dehydrated, and em-
bedded in the standard manner for TEM examina-tion.
Results
Examination of paraffin-embedded sections of kid-neys from experimental animals revealed bire-fringent, polycrystalline particles in the renal tubules,arranged in rosette or sheaf patterns characteristic ofcalcium oxalate. SEM examination showed two basic
configurations of crystals: dipyramidal CaOx dihy-drates and plate-like monoclinic CaOx monohy-drates, the latter being the most common type. Inultrathin sections, crystals appeared as holes in theplastic (Figure 1) or as shells filled with the plastic,depending on whether the crystals were removed byacid treatment or not. Even in TEM, one couldrecognize the rosette type arrangement and plate-likestructure of some crystals. Crystals appeared to bebounded by a membrane-like structure, which had anamorphous or finely granular material of variableelectron density deposited on it.
Associated with the crystals was a material that ap-
peared eosinophilic, amorphous, or granular by LMexamination, and tubular, fibrillar, or amorphous bySEM examination. TEM examination revealed thismaterial to be cellular debris composed of mem-
branous and vesicular material and cellular organellesin various stages of necrosis. Some of the necroticmaterial originated in proximal tubules, indicated byremnants of the brush border. Often a membranecould be identified around a mass of organelles, sug-
gesting that whole cells were sloughed into thetubular lumen, to become a part of the debris. Incrystal rosettes, the membranous and vesicularmaterial was typically present in the center.
Alterations in the structure of renal tubules were
limited to their crystal-containing portions, whichwere dilated and showed necrosis of their liningepithelia, whereas neighboring tubules withoutcrystals appeared normal, with neither dilation nor
necrosis. The intensity of necrosis, the size of CaOxparticles, their number, and their distribution withinthe inner medulla appeared to be dependent on thetime interval following NaOx injection. There ap-
peared to be a phase of necrosis associated with theappearance and growth of CaOx crystals, followedby their dislocation and a phase of renal recovery
(Table 1).One hour after NaOx injection, the CaOx crystals
were generally in small aggregates scattered diffuselythroughout the inner medulla (Figure 2) and re-
stricted to the tubular lumens. The tubular epi-thelium was generally intact, as was the urotheliumcovering the papillary tip. The surface of the papil-lary tip, with its slit-like openings of the ducts ofBellini, appeared normal. However, SEM examina-tion revealed occasional crystals of CaOx occludingthe ductal openings (Figure 3), where the crystals ap-
peared to exert pressure on the ductal epithelium,
Figure 1 -TEM of collecting duct 3 hours after injection. Crystals of CaOx (C) appear as holes and are attached to tubular basal lamina (B) byparacrystalline structures (X), which appear to extend into the interstitium (/). Tubular lumen contains cellular debris. Crystals appear to bebound on the luminal side by a membranelike entity (arrow). (x 24,840)
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Table 1 -Synopsis of Evolution of Medullary Changes
Location
Time Loop of Henle Collecting duct Papillary tip
1 hr Crystal aggregates small, present Epithelial intercellular spaces widened, Normal with occasional crystals in ductsin tubular lumen; epithelium spaces also present between epithelial of Bellinigenerally intact, occasional intra- basal lamina and basal cytoplasmiccellular edema membrane
3 hr Crystal aggregates small, in- Crystal aggregates larger, often com- Basically normal, with crystals in lumencreased intracellular edema, focal pletely occluding the tubular lumen, of ducts of Bellini, occasionally urothe-loss of epithelium epithelium of crystal-containing lium covering tip focally denuded
tubules either denuded or flattenedwith condensed cytoplasm, crystalsattached to the tubular extracellularbasal lamina
6 hr Crystal aggregates larger and still Some crystals intercellular, while Large aggregates of crystals in ducts ofin tubular lumens; loss of epithe- others intraluminal and still others Bellini, as well as their openings into thelium more pronounced attached to the exposed basal lamina renal pelvis, these openings are highly
of tubules; crystal aggregates in pe- deformed; urothelium covering the tip isripheral collecting ducts eroded often eroded; a number of crystalsthrough the epithelium to form lesions attached to the basal lamina of theon the papillary surface ductal epithelium as well as the urothe-
lium covering the tip
24 hr Fewer crystal aggregates, epithe- Fewer crystal aggregates, fewer areas Urothelium covering tip surface islium basically normal without of epithelial loss, epithelium still ulcerated and focally piled in 2, 3, or 4intracellular edema, areas of flattened but much more intact than layers; crystals present between epithe-epithelial loss fewer previous times lium and basal lamina in ducts of Bellini
48 hr Of crystals remaining in kidney Epithelium still flatter than normal; Urothelium still ulcerated, focally piledmost are in the loop; epithelium fewer crystal aggregates, which are up; slit-like openings of ducts of Belliniis basically intact smaller in size still difficult to identify
72 hr Crystals mostly in the loop; Almost normal, with few exceptions; Almost normal; urothelium covering theepithelium generally normal epithelial cells of normal height tip appears to have been reconstituted;
without intracellular edema or widen- slit-like openings of ducts of Bellini areing of intercellular spaces easily identified
168 and 336 hr Normal Normal Normal
causing cells lining the opening to separate from eachother. Transmission electron microscopic examina-tion showed intracellular edema in the cells of theepithelial lining of the thin limbs of Henle's loops, aswell as in those of collecting ducts. The epitheliumlining the limb occasionally appeared to be shearedoff the underlying basement membrane, thus exposedto the tubular contents. The intercellular spaces be-tween the collecting duct cells were often widened,although tight junctions remained intact. In the col-lecting duct, spaces also appeared between the basallamina and the basal cytoplasmic membrane of epi-thelial cells. In association with the crystals, therewas a small amount of membranous debris and ne-crotic organelles in the lumens.
Three hours after injection, the crystal aggregateswere larger and mostly located in the collecting ducts,many of which were occluded by crystals and cellulardebris. The epithelial changes were variable at thisstage. The crystal-containing tubules were extensivelydenuded of epithelium. Where the epithelium was in-
tact, the cells were either flattened, with condensedcytoplasm, or showed evidence of severe damage,with increased numbers of free ribosomes and poly-somes, swollen mitochondria, increased Golgi appa-ratus, and large lysosomes or phagosomes.
In those areas where epithelium was lost or thecells widely separated, crystals associated withcellular debris were seen lying against or attached tothe basal lamina. With TEM examination, organizedangular structures made of fibrillar, granular, andtubular material were usually seen between the basallamina and the layer of CaOx crystals and appearedto anchor the crystals to the tubules (Figure 1). Fre-quently these paracrystalline structures extended intothe interstitium, incorporating the basal lamina ofthe tubule and, often, that of the adjacent capillary(Figure 4). Cells were usually absent in such areas,though a thin layer of cytoplasm was present betweensome of these paracrystalline structures and thetubular basal lamina (Figure 4). Neighboring epi-thelial cells occasionally extended over the luminal
62 KHAN ET AL AJP * April 1982
CALCIUM OXALATE NEPHROLIrHIASIS 63
side of the crystalline complex. Similar angularparacrystalline configurations were also seen free inthe tubular lumen, where they appeared to incor-porate vesicles and multivesicular bodies fromdamaged cells (Figure 5), and fibrillar materialsimilar in appearance to that seen in the paracrystal-line structures was also present on the surface ofCaOx crystals lying free in the tubular lumen.On the papillary tip scattered collections of crystals
were seen between the lining urothelium and thebasal lamina. The papillary tip urothelium waspatchily denuded, leaving fragments of basalcytoplasmic membrane and exposing most of thebasal lamina. Generally, the epithelium of thepapillary tip and of the terminal ducts had a normalintracellular ultrastructure. Some of the intercellularspaces were enlarged and filled with electron-densegranular material with or without vesicular structuresembedded in it.At this time, TEM studies showed a progressive
loss of epithelium lining the limbs of the loop and apronounced intracellular edema.
Six hours after injection crystals of CaOx werepresent mainly as large aggregates near the papillarytip, loss of tubular epithelium was more pronounced,and there was more cellular debris in the tubularlumens. At the papillary tip, crystals could be seen inthe ostia of the ducts of Bellini (Figure 6) as theyopened into the urinary space. Layers of crystals ap-posed to the basal lamina of the ducts of Bellini andpapillary tip urothelium (Figure 6) were often associ-ated with loss of the urothelium, and sheets ofurothelial cells were seen in the pelvic space. WithSEM examination, the slit-like openings of the ductsof Bellini were highly deformed (Figure 7), and theurothelium at the tip was often eroded, revealing alayer of protruding crystals (Figure 7). Because of in-creased tubular necrosis, larger crystal aggregates,and occluded tubular lumens, it was now difficult todetermine the exact location of CaOx crystals by LMor SEM examination, but TEM examination clearlyshowed some crystals in the intercellular spaces oftubular epithelium, as well as forming layers in thevicinity of the tubular basal lamina, from which mostof the epithelial cells had apparently sloughed.Crystal aggregates in peripheral collecting ductsoften eroded the ductal epithelium and surfaceurothelium, resulting in the formation of crystallinemasses on the papillary surface (Figure 8).At 24 hours following NaOx injection, the amount
of crystals varied from animal to animal but ap-peared considerably less than earlier. These crystals
Figure 2-Rat renal papilla under polarized light, 1 hour after injec-tion.
were concentrated in the distal portion and the papil-lary tip. At this time, the collecting ducts were linedwith flattened epithelium. At the papillary tip, thecrystals were between the epithelium and the basallamina. The loss of urothelium seen in previousstages was no longer as evident, although free sheetsof urothelium were still seen in the renal pelvis. Theurothelium covering the papillary tip in areas wastwo to four layers thick. By SEM examination, theseareas of locally thickened epithelium appeared as epi-thelial folds on the surface of the papillary tip.By 48 hours there were fewer crystals scattered
throughout the inner medulla, and many of themwere in the thin limbs of the loop rather than in thecollecting ducts. The epithelial lining of the collectingduct was generally flatter than normal. An occasionalcrystal could still be seen in the opening of a ductof Bellini, but the layers of crystals seen previouslywere no longer present. At the papillary tip itself, thelining urothelium was intact and piled up focally intopapillary projections into the pelvic space. SEMshowed that the slit-like openings of the ducts ofBellini were still difficult to identify, and theepithelium was focally organized into folds.
Vol. 107 * No. 1
64 KHAN ET AL
s -- ** 's
K'
Figure 3-SEM of a papillary tip 1 hour after injection. Note the opening of a duct of Bellini occluded by CaOx crystals. Urothelium appears tobe mechanically disrupted (arrows). (x 1942)
Figure 4-TEM of the basal part of a collecting duct epithelium of a crystal-containing tubule. Tubular lumen is full with cellular debris con-sisting of degenerating organelles. Tubular epithelium appears disrupted and at places, as illustrated here, is represented only by a very thinlayer of cytoplasm (E). Unlabeled arrows point to the luminal membrane of epithelium. Paracrystalline structures (X) appear not only to incor-porate the basement membrane of the ductal epithelium (B1), but also of underlying capillary (B2). I = interstitium, A = capillary, C = CaOxcrystals. (x 20,240)
Figure 5-TEM of a paracrystalline structure (X) present in the collecting duct lumen, 3 hours after injection. Vesicles (V) from the cellulardebris appear to be incorporating in this structure. E = epithelium, N = nucleus. (x 33,000)
Seventy-two hours after injection, the few crystalsstill present were located mostly in the thin limbs ofHenle. The papillary tip epithelium appeared recon-stituted and was essentially intact, with an occasionalpiling up of epithelium in some spots, and the open-ings of the ducts of Bellini could be clearly identified(Figure 9). Occasional fragments of epithelium stillwere seen hanging from the papillary tip. TEM re-vealed absence of cytoplasmic intracellular edema.The cytoplasm appeared to be of normal electrondensity. Widening of the intercellular spaces in col-lecting ducts encountered at earlier times was not asfrequent. However, electron-dense granular materialwas still occasionally seen in the intercellular spacesin papillary collecting ducts.One hundred sixty-eight and 336 hours after NaOx
injection, only an occasional crystal or dilatation of acollecting duct was observed, and the papillary tipsurface was normal when viewed with the scanningelectron microscope.
Figure 6-Median longitudinal section through papillary tip, 6 hoursafter injection. Opening of a duct of Bellini (arrow) is occluded bycrystals of calcium oxalate and debris. Epithelium is denuded fromthe papillary tip and part of the duct exposing layer of CaOx crystals(arrowheads). At places, part of sloughed epithelium (E) is still pres-ent. (H&E, x 40) (With a photographic reduction of 23%)
66 KHAN ET AL AJP * April 1982
.0
-1 , ,.
Figure 7- SEM of the papillary tip 6 hours after injection showing the covering urothelium (E) being sloughed, exposing the underlying layer ofcrystals. (x 255) Compare with LM (Figure 6).
p
Figure 8-SEM of an ulcerated area on papillary surface 6 hours after injection, showing disrupted urothelium (E) and exposed crystals (C).(x 1950)
CALCIUM OXALATE NEPHROLITHIASIS 67
Figure 9-SEM of a papillary tip 72 hours after injection. The urothelium has been reconstituted. Ductal openings are again identifiable (ar-rows). Epithelium (E) appears to be hanging through a ductal opening. At some places, urothelium appears to be migrating laterally (arrow-heads) from warty excrescences on the tip surface. (x 277)
Discussion
The question of whether CaOx crystals are pri-marily intra- or extracellular is believed to be of im-portance in the pathogenesis of urolithiasis. Ourearlier studies have shown that crystals of CaOx inthe renal cortex resulting from an injection of NaOxare primarily intratubular. 12.1.1 8 In other studies ofexperimental CaOx nephrolithiasis, CaOx microlithshave been shown to be intraluminal in proximaltubules, as, for example, in rats fed an oxalic-acid-containing diet." However, in rats, administra-tion of ethylene glycol produces deposits of CaOx inboth intraluminal20 and intracellular locations,2' andvitamin-B6-deficiency-induced CaOx microliths havebeen variously described as intraluminal,22 intra-tubular near basal lamina,23 or interstitial.24The results of the present study indicate that
following NaOx injection, intratubular crystals ofCaOx are present first in the renal cortex and then inthe medulla, including the papilla. Initially thecrystals are intraluminal, but later they are alsolocated intercellularly, as well as between the tubularcells and the basal lamina.
In addition, the appearance of CaOx crystals inrenal tubules following NaOx injection is associated
with necrosis of tubular cells, which results in ex-posure of the tubular basal lamina and formation ofluminal cellular debris. The mechanism of the tubu-lar necrosis is not known. However, because tubularnecrosis is limited to areas of tubules containingcrystals, we speculate that a causal relationship existsbetween the crystals and the cellular necrosis.As yet there is no direct evidence that CaOx crystals
are toxic to renal epithelial cells, but such crystals docause cytolysis of polymorphonuclear leukocytes fol-lowing phagocytosis and of erythrocytes secondaryto external membranolytic mechanisms.25 A causalrelationship between CaOx crystals and necrosis ofmyofibrils in uremic patients has also been suggested.26The crystal aggregates may be destructive to renal
epithelium because they are large and irregular and,as demonstrated by our SEM studies, appear to me-chanically disrupt the epithelium. This may be a gen-eral phenomenon in nephrolithiasis, since we haveobserved it in oxamide lithiasis as well.2" Anotherpossible cause of epithelial necrosis may be brief ex-posure of the cells to a relatively high oxalate ionconcentration, which may be a general cytotoxin. Inaddition, transient renal ischemia may play a role,because ischemia has been shown to cause papillarynecrosis, 8-30 and NaOx, as used in these experiments,
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68 KHAN ET AL AJP * April 1982
results in aortic constriction and edema in the peri-toneal cavity.17
In our experiments the renal papilla and innermedulla underwent changes in two phases. An initialphase of crystal formation, aggregation, and growth,which was associated with cellular necrosis, was fol-lowed by a phase of crystal dislocation, attachmentto basal lamina, cellular recovery, and reepithelial-ization of the denuded areas. The pattern of progres-sive distortion of the papillary tip followed by recov-ery is similar to the pattern of papillary changesobserved by us in oxamide urolithiasis" and by Cup-page and Tate28 after bromoethylamine hydrobro-mide administration.For the first hour after injection, crystals of CaOx
appeared free in tubular lumens, but by 3 hours, anumber were attached to the tubular basal lamina byunusual angular paracrystalline structures that mergedimperceptibly with the tubular basal lamina. Thecomposition of these paracrystalline structures is un-known, but they may represent a coating on the crys-tal surface formed by the deposition of cellular debrisand of urinary proteins. Calcium oxalate crystalshave an affinity for proteins,31 and the fact that thesestructures were observed only in medullary parts ofthe nephron suggests that Tamm-Horsfall urinarymucoprotein may play a role. The idea is attractive,because Tamm-Horsfall protein has been identifiedin a variety of human kidney diseases, where it may bedischarged following tubular disruption,3233 and hasbeen detected in the matrix of sheep struvite calculi.34
Because of the observations reported above, aswell as those in our earlier studies,1,2,17,18 we suggestthat the following events occur in NaOx-inducedCaOx nephrolithiasis. Calcium oxalate crystals areformed intraluminally in proximal tubules, wheresome crystals attach to the proximal tubular epithe-lial cells, but are released because of the loss of brushborder2 or necrosis of the cells. The crystals becomecoated with cellular debris and urinary proteins,which facilitate their aggregation. Some crystallineaggregates coming in contact with an exposed base-ment membrane become attached and act as a nidusfor the formation of microliths, which grow by fur-ther addition of crystals, finally causing obstructionof the nephron. Alternatively, some crystals mayform in the medulla at the collecting duct basal lam-ina or in the intercellular spaces. Obstruction ofducts located near the medullary surface results inerosion of the overlying ductal epithelium and theurothelium, forming a lesion comparable to a Ran-dall Type I plaque, whereas obstruction at the papil-lary tip results in "intratubular inspissation" of termi-nal collecting ducts, forming a lesion comparable to a
Randall Type II plaque. In fact, Prien35 has describeda similar production of Type II lesions by experimen-tal induction of hyperoxaluria in pyridoxine-deficientrats. During the phase of recovery, a portion of thepapillary tip is sloughed, a new epithelium is reconsti-tuted, and finally the papilla appears normal. Crystalsmay persist for long periods, and we have observedcrystals in the renal parenchyma for up to 2 weeks,particularly in the loops of Henle. When large crystalaggregates persist in the narrow thin limbs, they maybe picked up by lymphatics, as suggested by Carr,36although we see no evidence for this in our material.
It has been suggested that initiation of stone dis-ease in the upper urinary tract is most often the resultof a fixed particle mechanism.37 Here we have shownone mechanism by which such fixation can be accom-plished. We have illustrated the similarities betweenRandall's plaques found on human renal papillae38and the lesions formed on the papillary tip andmedullary surface of rat kidneys. However, experi-mental difficulties prevent us from making a directcomparison of the sequence of morphologic changesthat occur in experimentally induced CaOx nephroli-thiasis with the sequence of morphologic changesthat occur in human "idiopathic" CaOx nephroli-thiasis. We believe it is likely that some of the eventsin the pathogenesis of upper urinary tract stone dis-ease will prove to be similar to those in this model ofexperimental nephrolithiasis.
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AcknowledgmentsThe authors wish to thank Dr. Henry Aldrich, of Depart-
ment of Microbiology and Cell Science, University of Flor-ida, for the use of his electron microscope facility, and Dr.Benjamin Trump, University of Maryland, for his com-ments on selected micrographs. Mr. Jack Konicek providedthe excellent technical assistance.
