Analysis of microcracks caused by drop shatter testing of porcine kidneys

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Ann Anat 191 (2009) 294—308

RESEARCH ARTICLE

Analysis of microcracks caused by drop shattertesting of porcine kidneys

Zbynek Tonara,b,�, Jirı Janacekc, Lukas Nedorosta, Robert Grilld,Vaclav Bacae, Frantisek Zat’uraf

aDepartment of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague, Karlovarska 48,301 66 Pilsen, Czech RepublicbDepartment of Mechanics, Faculty of Applied Sciences, University of West Bohemia, Univerzitnı 8, 306 14 Pilsen,Czech RepubliccInstitute of Physiology, The Academy of Sciences of the Czech Republic, Vıdenska 1083, 142 20 Prague 4, CzechRepublicdUrology Clinics, University Hospital Kralovske Vinohrady, Srobarova 50, 100 34 Prague 10, Czech RepubliceDepartment of Anatomy, Third Faculty of Medicine, Charles University in Prague, Ruska 87, 100 00 Prague 10, CzechRepublicfUrology Clinics, University Hospital in Olomouc, I.P. Pavlova 6, 775 20 Olomouc, Czech Republic

Received 5 May 2008; received in revised form 8 December 2008; accepted 9 February 2009

KEYWORDSExperimental trau-ma;Histology;Kidney;Microcracks;Rupture;Tubules;Stereology

SummaryAlthough kidney trauma is a relatively common injury, its microscopic biomechanicsare poorly understood. Experimental low-grade trauma in pig kidneys was studiedusing optical microscopy. We observed ruptures in the cortex as well as in themedulla. Both parts of the renal parenchyma were damaged, even in areas of thekidneys that were free of macroscopic cracks on the surface. To determine whichconstituents of the renal cortex and medulla, i.e. tubular parts of the nephron or theinterstitial connective tissue, were less resistant to injury during the drop shattertest, we applied a simple stereological method to discriminate between random andtissue-specific rupture propagation. The ruptures propagated predominantly throughthe interstitial connective tissue of the renal cortex and medulla. The volumefraction of the tubules assessed by the Cavalieri principle was 90.4% within the renalcortex and 52.4% within the medulla. The most frequently affected blood vesselswere the arcuate and interlobular veins, followed by the arcuate and interlobulararteries. No disruptions of the renal calyces were found.& 2009 Elsevier GmbH. All rights reserved.

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0940-9602/$ - see front matter & 2009 Elsevier GmbH. All rights reserved.doi:10.1016/j.aanat.2009.02.005

�Corresponding author at: Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague,Karlovarska 48, 301 66 Pilsen, Czech Republic. Tel.: +42 0377593320, +42 0607818614; fax: +42 0377593329.

E-mail address: [email protected] (Z. Tonar).


Introduction

The renal cortex (C) consists of renal corpusclesand closely packed proximal and distal tubulessurrounded by delicate connective tissue withblood and lymph vessels. The cortex can be dividedinto outer and inner (juxtamedullary) zones. Theinner zone is demarcated from the medulla bytangential arcuate arteries and veins that branchinto interlobular arteries. A thin layer of subcortexsurrounds the arcuate vessels on the medullary side(Standring et al., 2005). The renal medulla (M)contains thick and thin limbs of the loops of Henleand vasa recta. Collecting ducts originate in thecortical medullary rays, run through the medullaand finally open into wider papillary ducts whichopen into papillae. The propensity of the kidneysfor traumatic rupture is believed to be related tothe presence of a rich vascular bed.

Cracks in the renal parenchyma can result fromtrauma, e.g. during a traffic accident, or they maybe caused by simple falls. The latter often occur inelderly people with impaired mobility or coordina-tion. Symptoms may vary from temporal micro-scopic haematuria with negative results on kidneyultrasonography to severe deterioration of renalfunction and massive bleeding. In this paper, wefocus on experimental low-grade kidney trauma(Moore et al., 1989). Although it is a relativelycommon injury, its microscopic biomechanics arepoorly understood. To our knowledge, there havebeen no reports quantitatively describing thehistological traumatic changes to the kidney cortexor medulla.

Urogenital trauma is reported to account for 10%(Schmitt and Snedeker, 2006a) to 20% (Snedekeret al., 2005) of all abdominal injuries. Statistically,the risk of renal pathology is characterised by theassociation of monotrauma, macroscopic haema-turia and low impact velocity (Schmidlin et al.,1998). Clinical aspects of blunt abdominal traumawith kidney injury were published by Husmannet al. (1993), and systematically reviewed bySantucci and Fisher (2005). It is known thatabnormal kidneys are more vulnerable to injury(Schmidlin et al., 1998; Ceylan et al., 2003; seeSantucci and Fisher, 2005 for a meta-analysis ofincidence and types of abnormal kidneys and theirinjury risk). A computer-simulated kidney traumamodel revealed that incompressible objects filledwith a liquid (e.g., cysts or hydronephric renalpelvis) could amplify the force of the traumaimpact (Schmidlin et al., 1996). In children,the kidney is at greater risk because of itsproportionally larger size and limited perirenal fatprotection (Ceylan et al., 2003). Together with

the development of imaging algorithms andtechniques (for the validity of computerised tomo-graphy of renal trauma see Bschleipfer et al.,2003), numerical models with predictivecapabilities are evolving as parts of expertsystems for preliminary clinical diagnosis. Develop-ment of useful computer models requires informa-tion on the microstructural changes occurringduring renal injury. Accurate biomechanical inputdata required by such models are not sufficientlyavailable.

Most recent papers dealing with blunt kidneyinjury either reviewed medical records or per-formed impact experiments using porcine kidneysas model organs. Using density measurements,compression and tensile tests, as well as sheartests, the material properties of fresh pig kidneyswere described and their elastic and inelasticmaterial behaviour characterised by Farshad etal. (1999). Schmitt and Snedeker (2006a) deter-mined the biomechanical response of whole,perfused porcine kidneys to blunt impact anddemonstrated that injury was best predicted byimpact energy. Bschleipfer et al. (2002) demon-strated in porcine kidneys subjected to dropimpactor injury, that the first lesion originated inthe region between the renal pelvis and cortex.When the pelvis was filled with fluid (bothexperimentally as well as in vivo), it acted as anincompressible support that was assumed to changeits shape after submission to more than about 4 J ofapplied energy. The poles of the kidney remainedthe least damaged areas. The authors (Bschleipferet al., 2002) also evaluated the force distributionand the timing of biomechanical parameters untilthe first lesions appeared. Other impact tests wereperformed on whole perfused porcine kidneys(Snedeker et al., 2005) in order to estimate thestrain energy density leading to rupture. However,in all the papers mentioned above, the injurieswere only macroscopically assessed; none utilisedmicroscopic analysis.

As previously published (Grill and Zat’ura, 2006),58% of our patients (n ¼ 228) treated for bluntkidney injury were diagnosed as grade I or IIaccording to the renal injury scale of the AmericanAssociation for Surgery in Trauma (Moore et al.,1989). In general, these renal injuries rarelyrequire surgical intervention, in contrast to mostinjuries of grade III and higher. According to ourexperience, the first morphological posttraumaticchange in the kidney is interstitial bleeding in thekidney parenchyma. Diagnosis of some of smallercracks (i.e. under 5mm) without conspicuousbleeding remains beyond the limits of ultrasono-graphy.

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Histology of experimental blunt renal trauma 295


In biomechanical experiments with porcine kid-neys, histology can provide data that are currentlymissing from the literature. This might help todescribe the resistance of kidney microstructuresto shear stress, even in an experimental design thatneglects the anatomical boundary conditions oforgans surrounding the kidney in vivo. Therefore,the first aim of the present study was to answer thefollowing questions:

1. Which of the constituents of the renal cortexand the medulla offer less resistance during adrop shatter test simulating a low-grade (I and II)blunt kidney injury? Are these the tubular partsof the nephron or is it the interstitial connectivetissue?

2. What kind of microscopic changes can resultfrom low-grade blunt kidney injury? Are thereother acute traumatic changes (e.g. vascularinjuries) even in those parts of the renalparenchyma which are free of macroscopiccracks?

The second aim of this study has been to supplydata on the volume fraction of renal tubules for anovel non-trivial computer model of the kidneythat describes the parenchyma as a porous mediumconsisting of tubular structures interpenetratingthe connective tissue matrix (Cimrman and Rohan,2007). Therefore, we estimated the volume frac-tion of the tubular part of the nephron within therenal cortex and medulla.

Material and methods

Mechanical experiment

Kidneys (n ¼ 33) were removed from freshlyslaughtered domestic pigs (m ¼ 100–120 kg). Thesex of the animals was unknown. However, themale pigs were bred in large-capacity animalhouses in the Czech Republic and had beencastrated as juvenile animals, which reduced theirsexual dimorphism. Each kidney had a shortsegment of renal artery, renal vein, and ureterattached. The experiment was performed withorgans taken from cadavers, and the animals wereslaughtered in the slaughter-house according toArticle 26 of the European Convention for theProtection of Vertebrate Animals used for Experi-mental and other Scientific Purposes (ETS No. 123),valid in the Czech Republic. The study protocol wasapproved by the local research ethics committee onMay 30, 2005 (University Hospital in Olomouc). The

interval of warm ischemia did not exceed 2 h. Toprevent dehydration, the organs were stored in a0.85% saline solution chilled at 0 1C until testingoccurred (the testing was performed the sameday). In order to detect any traumatic changes orabnormalities present before the mechanical ex-periment, kidneys were examined via ultrasono-graphy, and those that did not pass thisexamination (n ¼ 4) were excluded from the study(the results of ultrasonography and study ofcorrosion cast preparations of the blood vesselswill be published separately in a clinically orientedpaper). After exsanguination of the kidneys, flex-ible cannulae were inserted into the renal arteryand ureter and fixed by ligation. The organs wereperfused with physiological solution, stored in amixture of ice and water and transported to thelaboratory. Before perfusion, the weight of thekidneys was 167765 g; after perfusion, the weightwas 244763 g (mean7standard deviation). Theweight of the saline present in the vascular bedand pelvis was thus 7677 g; the perfusion in-creased the weight of the exsanguinated kidneysby 51714%.

The mechanical experiment was performed in30 kidneys on the same day as the preparation ofthe organs and consisted of a free fall of thekidneys on their dorsal surface at room tempera-ture. The perirenal fat was removed prior to theexperiment. The dorsal surface of the kidney hit ametal plate at a velocity of 5m s�1. The firm metalplate simulated the smooth and rigid surface of theribs. The calculation of the impact velocity v wasbased on the average height of the centre of gravityof an adult person (h ¼ 127 cm) and on theassumption of free fall, v ¼

ffiffiffiffiffiffiffiffi2gh

p, where g is the

standard gravity acceleration (9.81m s�2). Duringthe drop shatter test, the pressures of thephysiological solution within the arterial systemand the pelvis of the kidney were maintained at16 and 1.47 kPa, respectively. The latter valuecorresponds to the normal pressure within thepelvis according to Whitaker’s test (Whitaker andCuckow, 1994). We did not evaluate the effect ofimpact energy, which has already been sufficientlyinvestigated (Schmitt and Snedeker, 2006a, 2006b).The macroscopic results of the drop shatter test(Grill and Zat’ura, 2006) showed 5–9 ruptures(6.571.6, mean7SD) of the parenchyma on thesurface of each kidney. The length of theseruptures ranged from 0.5 to 22mm. There werealso 1–4 ruptures of the renal capsule of eachkidney (2.371.1, mean7SD), the length of whichranged from 5 to 40mm. The localization of themacroscopic ruptures of the parenchyma matchedthe localization of the ruptures of the capsule

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Z. Tonar et al.296


(at least in part) 33% of the time, while 66% of theruptures of the parenchyma occurred below anintact capsule.

Sampling

Three kidneys were randomly selected for me-chanical experimentation and labelled as kidneynos. 1–3. The three remaining kidneys underwentall processing described above (i.e., removal ofperirenal fat and perfusion) but were not dropped,thus serving as negative controls and were labelledas kidney nos. 4–6. Kidney nos. 1–6 underwent thesame histological sampling and analyses. Six tissueblocks were extracted from each kidney: two fromthe ventral surface, two from the dorsal surfaceand two from the caudal pole. Several parts ofthe surface were free of ruptures; in such cases,the position of the tissue block within each region(ventral/dorsal) was also random. As the caudal

pole of the kidney suffered no apparent ruptures,it was considered the least affected region.A 6mm-thick slab, sampled in a random mannerfrom each tissue block, was rotated around avertical axis perpendicular to the kidneycapsule and embedded in paraffin. From the firsthistological section containing a profile of therupture, a series of 200 histological sectionswere cut at a thickness of 5 mm. In this manner,a 1mm-thick tissue sample was sectioned exhaus-tively. The sampling procedure is presented inFigure 1.

All segments of the ruptures and all parts of therupture-free segments of the kidneys were giventhe same chance for selection in the study.We considered this procedure capable of providingus with a sample that would which enable us tostudy the course of rupture propagation. Alto-gether, we analysed 36 tissue blocks including alllayers of the kidney, i.e. the fibrous capsule, thecortex, the medulla, and the adjacent fat within

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Figure 1. Sampling of tissue blocks and slabs. From each kidney, six tissue blocks were extracted from three regions:two blocks from the ventral surface, two blocks from the dorsal surface, and two blocks from the caudal pole. In casesof superficial cracks (length of 0.5–22mm), the whole tissue block containing the crack was extracted from the relevantregion with �3mm borders of macroscopically intact tissue. If the region contained no apparent rupture, e.g. in thecaudal regions of all kidneys, the coordinates of the lateral and cranial origin of the block [h;w] were selected as themaximal height and width of the kidney [hmax;wmax] multiplied in a spreadsheet by a random number between 0 and 1.If the square with the cranial and lateral corner [h;w] was not completely within the irregular borders of the regionfrom which the block was to be sampled, the calculation was repeated. From each of the extracted blocks, a 6mm-thick slab was sampled. The position of the slab to be sampled (x) was calculated as the length of the block (xmax)multiplied by a random number between 0 and 1. The 6mm-thick slab was rotated around a vertical axis (perpendicularto kidney capsule) at a random angle j and embedded into paraffin. The section plane was perpendicular to the kidneysurface. From the first histological section containing a profile of the rupture, a series of 200 histological sections withthickness of 5 mm was sectioned, thus a �1mm-thick tissue sample was sectioned exhaustively. All sections were savedand stained, and every 20th section was used for quantitative assessment of the ruptures. In order to minimise themanipulation of the tissue sample, only the sampled 6mm slab was cut physically; the positions of the other slabs in thescheme were virtual.



the central renal sinus and the wall of the renalcalyces and pelvis.

Assessment of microcracks

Each of the randomly selected tissue samples wasprocessed by common paraffin technique and cutinto a series of 200 slices at a thickness of 5 mm. Thesection plane of all blocks was perpendicular to thekidney surface, but randomly rotated around a‘‘vertical axis’’ perpendicular to the kidney cap-sule. These sections were stained with haematox-ylin–eosin (HE) and Mallory trichrome, the latterproviding sufficient contrast between the wall ofthe nephron and the connective tissue. Usingsystematic uniform random sampling, we selected10 slices from each of the tissue blocks for furtherlight microscopy analysis. We used an OlympusBX51 microscope with objectives UPlanSApo10NA ¼ 0.40, 20 NA ¼ 0.75, and 40NA ¼ 0.90.The total number of micrographs analysed was180. The typical morphology of a cortical rupture isshown in Figure 2A.

To study the spatial relationship of microcracksand kidney tubules, we compared the estimatedvalue of the intersection intensity of tubule profilesalong the crack P0L with its theoretical value PL,calculated under assumption of complete spatialindependence of the cracks on tubule profile. Theintersection density of the tubule profiles wasdefined as follows:

PL ¼pl

(1)

in which p was the number of the intersectionsalong a crack of length l.

The intensity of intersections of rupture profileswith the inner circumference of renal corticaltubules P0L was estimated by dividing the realnumber of these intersections (p0) counted in thesections (Figure 2B) by the maximal projection

length of the profiles (l). We used the maximalprojection length of the crack as a lower bound ofthe real length. We estimated the maximal projec-tion length of the cracks l (Figure 2B) as thedistance between the beginning and the end ofthe rupture profile (Ellipse software, Line tool).The endpoints of the maximal projection length ofa crack were defined as the furthest boundariesbetween crack profile and intact tissue (in the caseof superficial cracks, the thickness of the kidneyfibrous capsule was not included). We assessed themaximal projection length l and the number ofdisrupted tubules p0 for both edges of the ruptureand calculated an average value for the lengths andcounts.

The theoretical intersection intensity under theassumption of independence of the cracks andtubule profiles was calculated as the intersectionintensity of a randomly oriented line (i.e. theprofile of a rupture) with the tubules (Stoyan et al.,1995) as follows:

PL ¼2pLA (2)

in which LA was the length density (intensity ofplanar fibre process according to Stoyan et al.,1995) of the profiles of the inner circumference ofthe tubules. In other words, the theoreticalintensity of the intersection of the tubule profileby a randomly oriented linear structure in a two-dimensional micrograph was proportional to thelength density of the tubule. The constant ofproportionality in this relationship is given by theprobability of the selection of the line, whichequals the mean height of a randomly orientedabscissa (2/p in two dimensions; the theoreticalbackground can be found in Stoyan et al., 1995 orRuss and Dehoff, 2001).

For each kidney, we assessed the length densityLA of the profiles of the inner circumference of thecortical or medullar tubules, respectively, as

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Figure 2. (A) Rupture running through the renal cortex. Scale bar 200 mm, HE stain. (B) Counting of the rupturedtubules (green asterisks). On the left, the edge of the rupture is highlighted in orange. On the right, the maximalprojection length l is drawn as a blue line, demonstrating how it was measured for the study.

Z. Tonar et al.298


follows:

LA ¼LA

(3)

in which LA is the length density (intensity of planarfibre process according to Stoyan et al., 1995), L isthe length of the inner circumference of therelevant tubules, and A is the area of the referencespace. To estimate the L and A parameters,we used the LineSystem module of Ellipse software(ViDiTo, Kosice, Slovakia). L was estimated bycounting the intersections between circular arcspositioned randomly on the micrographs and theinner tubular profiles (Figure 3). A was estimatedwith a point grid. The minimum number of theseintersections always exceeded 200 for each seriesof 10 sections. We assessed LA for each tissue blockof each kidney (separately for cortex and medulla).

Statistical hypotheses

To be rejected in order to confirm a preferredtissue constituent for rupture propagation:

Hypothesis H0A. The intensity P0L of countedintersections of rupture profiles with the innercircumference of renal cortical tubules does notdiffer from the predicted value PL.

Hypothesis H0B. The intensity P0L of countedintersections of rupture profiles with the innercircumference of renal medullar tubules does notdiffer from the predicted value PL.

The H0A hypothesis claims that the rupturepropagates randomly through the renal cortex andprefers neither tubules nor interstitial connectivetissue, and the H0B hypothesis claims that therupture propagates randomly through the renal

medulla and prefers neither tubules nor interstitialconnective tissue. If the actual propagation of theruptures through the renal cortex and the renalmedulla was random, then Hypotheses H0A andH0B, respectively, would be valid and P0LEPL.A significant difference between the predicted PLand the actually observed P0L would indicate thatthe rupture ran predominantly through the tubules(P0L4PL) or through the connective tissue (P0LoPL).Differences between P0L experimental data and PLvalues derived from LA were assessed using the signtest (Statistica Base 7.1, StatSoft, Inc., Tulsa, OK,USA). The results for all blocks in one kidney(separated for cortex and medulla) were pooled asone sample.

Volume fraction of tubules

The Cavalieri principle implemented in thePointGrid module of Ellipse software was used forestimation of the volume of tubules within thereference volume of the cortex and the medulla asfollows:

estV ¼ TðA1 þ A2 þ � � � þ AmÞ (4)

in which estV is the Cavalieri volume estimator,T ¼ 100 mm is the distance between two selectedsections, Ai is the area of tubular profiles assessedwith the point-grid method in the ith section, andm ¼ 10. The minimum number of counted pointshitting either the tubular epithelium or the lumenof the tubules always exceeded 200 for each of theseries of 10 sections. The volume fraction ofcortical tubules in the cortex VV(tubules,cortex)and the volume fraction of medullary tubuleswithin the medulla VV(medulla,cortex) was thenassessed as follows:

VV ðtubules; cortexÞ ¼estVtubules

estVcortex,

VV ðtubules;medullaÞ ¼estVtubules

estVmedulla. (5)

Results

Experimental samples

The low-grade blunt kidney injury in our experi-ment produced cracks of the renal parenchyma inboth cortex and in the medulla, as well as rupturesof blood vessels in kidney nos. 1–3. Most of theruptures of the renal cortex started in the fibrouscapsule and propagated into the superficial part of

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Figure 3. Circular arcs contacting the profiles of theinner circumference of the renal tubules were used toassess the two-dimensional length density of the profilesLA.



the renal cortex (Figure 4A), sometimes withextensive branching. Other ruptures did not showany connection with the surface (Figure 4B), andwere thus present in samples that did not showruptures prior to the microscopic analysis. Rupturesof the cortex were present in half of the caudalpoles, irrespective of the absence of apparentruptures on the surface of these specimens. Nodamaged glomeruli were found. However, theparietal layer of Bowman’s capsule was disrupted(Figure 5B).

In the medulla of kidney nos. 1–3, ruptures alsooccurred either simultaneously with the corticalruptures or without any relationship to them(Figures 4C and D). All medullary microcracks werefound near the apices of the renal pyramids, i.e. in

the vicinity of the renal sinus. No disruptions of therenal calyces or of the adipose tissue in the renalsinus were found.

We tracked 10 ruptured tubules (five in thecortex, five in the medulla) in a series ofconsecutive sections. We were able to track thecracks in series of no more than nine consecutive5 mm-thick sections, corresponding to a distance of45 mm.

In the cortex, occasional ruptures of the arcuatevessels and their branches (the interlobular ar-teries) were found, even in samples withoutruptures apparent on the kidney surface. Arcuatearteries and veins were identified by their diameterand tangential position near the cortico-medullaryborder (Figure 5). They were surrounded by a thin

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Figure 4. (A) Most ruptures of the renal cortex started in the fibrous capsule and propagated into the superficial part ofrenal cortex, sometimes with an extensive branching. (B) This rupture was hidden deep in the cortex and was notconnected to the surface (i.e. it was not visible prior to microscopic analysis). Not only the interstitial tissue, but alsomany proximal and distal tubules were disrupted. (C) In the medulla, ruptures occurred either simultaneously withcortical ruptures or without any relationship to them. The fibrous renal capsule and the renal sinus are located in thelower part of the micrograph. There were no macroscopic ruptures on the surface. (D) All medullary microcracks werefound near the apices of the renal pyramids (in the vicinity of the renal sinus). No disruption of the renal calyces wasfound. Scale bars 200 mm (A, B), 180 mm (C), and 100 mm (D); Mallory (A–C) and HE (D) stain.

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layer of subcortex on the medullary side. Rupturedcapillaries were not observed in the cortex. In themedulla, ruptures of capillaries were not excludedbecause it was impossible to discriminate some ofthe capillaries from some of the thin segments ofthe Henle’s loops.

Control samples

No damage was found in the tissue blockssampled from kidney nos. 4 and 6. In one of thesamples taken from kidney no. 5, two ruptureswere found in the cortex, but unlike the ruptures inkidneys that were subjected to the drop shattertest, these ruptures could not be tracked inadjacent serial sections as they only occurred inisolated sections.

Quantitative analysis of the ruptures

Quantification of all ruptures is summarised inTables 1 and 2. The results of the sign testcomparing the values of P0L with PL (Table 2)allowed us to reject both Hypothesis H0A and H0B.The values of intensity P0L of the counted intersec-tions of rupture profiles with the inner circumfer-ence of both cortical and medullar tubules weresignificantly different from the predicted values PL.The ruptures thus did not propagate randomlythrough the renal cortex and medulla. In bothlayers, they predominantly ran through the fineinterstitial connective tissue. This connectivetissue was less mechanically resistant than tubulesand glomeruli during the drop shatter test, which

simulated low-grade blunt kidney injury. Thecontrol samples only contained infrequent micro-cracks. The volume fraction of the tubules withinthe cortex and within the medulla are summarisedin Table 3.

Discussion

Applicability of the porcine model to humans

When performing impact experiments on softorgans, a number of experimental limitationsshould always be considered. Kidneys isolated atroom temperature behave differently than kidneysin vivo. These tests were performed on porcinekidneys, and caution must be applied when inter-preting these results with regard to human safety.Fresh human kidney tissues, and particularly wholeorgans, are extremely difficult to obtain in quan-tities sufficient for thorough experimentation. Mostkidneys removed from human donors are used fortransplantation to recipients with renal failure.Due to the shortage of human tissues, animalmodels have been used in impact biomechanicsresearch (for a thorough review of animal impactstudies, see Nahum and Melvin, 2001). The porcinekidney is both anatomically and physiologicallysimilar to the human kidney; therefore, the porcinekidney can be substituted for the human kidney inbiomechanical tests. A general comparison ofthoracic and abdominal blunt insults in validatedporcine models and human models was performedby Shen et al. (2008). However, it has been

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Figure 5. (A) Rupture of two profiles of arcuate arteries between the superficial and deep renal cortex. The connectivetissue of adventitia and some of the tubules are damaged as well. No apparent rupture was found on the surface of thistissue block. (B) Rupture of the wall of an arcuate artery (left), arcuate vein (upper right vessel), and surroundingconnective tissue and tubules. The surface of the specimen was ruptured as well. Scale bars 200 mm; Mallory (A) and HE(B) stain.



demonstrated that the porcine kidney parenchymais more robust than the corresponding humantissue. When compared to the human kidney, theporcine kidney can withstand nearly 35% moredeformation energy before it fails (Snedeker et al.,2005). However, Schmitt et al. (2005) showed in anexperimental study with a pendulum impact devicethat porcine kidneys exhibit both similar types andlocations of injuries in response to identical bluntimpacts. Therefore, it is likely that if the experi-ment described in this paper was performed withhuman kidneys, more pronounced ruptures wouldhave occurred, but in a similar pattern. In cadaver

impact experiments at 4.7m s�1, Snedeker et al.(2007) observed no kidney injuries; however,impact tests performed with cadavers rather thanwith isolated kidneys might be affected by theprotection of the kidney by the abdominal wall,perirenal fat, and other surrounding organs.

The average dimensions of the human kidney are�10 cm in length, 6 cm in breadth, and 3 cm inthe antero-posterior dimension (the left kidney canbe 1.5 cm longer than the right). The averageweight is �150 g in men and 135 g in women(Standring et al., 2005). The superior portion ofthe kidneys is level with the upper border of the

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Table 1. List of samples and measured data.

Sample Rupture LA (mm�1) l (mm) p0 p

Kidney no. 1V1 C 0.10170.005 6127143 2.0 38.9V2 C 0.10270.005 5087109 2.6 33.0D1 C 0.10970.009 583793 3.1 40.2D2 C 0.08970.005 6267111 5.8 35.3D1 M 0.09770.005 6317117 6.2 39.0D2 M 0.10570.011 666734 5.1 44.5Ca1 – – – – –

Ca2 – – – – –

Kidney no. 2V1 C 0.10570.004 224762 2.3 15.0V2 C 0.10770.005 364753 3.8 24.7D1 C 0.10670.005 335759 0.5 22.6D2 C 0.11270.005 369739 1.0 26.2Ca1 C 0.09970.004 165760 0.4 10.4Ca2 C 0.09570.004 301799 0.6 18.2V1 M 0.13070.003 476782 6.6 39.5

Kidney no. 3V1 C 0.13070.006 365746 1.6 30.2V2 C 0.13070.022 351720 2.4 29.0D1 C 0.12970.006 418782 6.6 34.2D2 C 0.12870.005 370795 8.8 30.2Ca1 – – – – –

Ca2 C 0.12770.005 28722 0.1 2.3D2 M 0.22970.011 1487108 4.9 21.5

Kidney no. 4V1, V2, D1, D2, Ca1, Ca2 – – – – –

Kidney no. 5V2 C 0.10170.004 15173 3.0 9.7V1, D1, D2, Ca1, Ca2 – – – – –

Kidney no. 6V1, V2, D1, D2, Ca1, Ca2 – – – – –

Average values7standard deviations (SD) of series of 10 slices: LA, length density of the inner circumference of the tubules; l, themaximum projection length of the rupture; p’, actual number of intersections of the inner circumference of ruptured tubules along themaximal projection length of the crack l; p, hypothetical number of intersections of ruptures with the tubules as calculated with Eqs.(1) and (2). Kidney nos. 1–6 indicates the number of the kidney studies; V1-2, D1-2, and Ca1-2 indicate ventral, dorsal, and caudallocation and the number of the sample; C and M indicate cortical and medullar ruptures, respectively;– indicates ‘‘not found’’ or ‘‘notdefined’’.

Z. Tonar et al.302


12th thoracic vertebra, and the inferior portionwith the third lumbar vertebra. The right kidney isusually slightly inferior to the left. Although theporcine kidneys used in our experiment weresomewhat heavier (mean weight 167 g) than humankidneys, their dimensions were quite similar to theaverage human kidney. Compared with humankidneys, the pig kidney is somewhat compressedin the dorso-ventral direction, but elongated in thecranio-caudal direction. Unlike in man, the left andright kidneys are in nearly the same position withrespect to the first four lumbar vertebrae, with theright kidney often extending more cranially thanthe left kidney (Schummer and Vollmerhaus, 1995).While the shape and dimensions of porcine kidneysare quite similar to those in man, the anatomicaland mechanical boundary relationships are not.

Biologists or physicians not involved in biome-chanical experiments occasionally object to thetesting of kidneys isolated from their naturalanatomical environment. To prevent confusion,

we would like to emphasise that one of the keyconcepts of simulations in biomechanics is thedifference between the material properties of anorgan and its boundary conditions. Although theorgan properties are expected to be comparablebetween porcine and human kidneys, the boundaryconditions can differ substantially according to theanatomy of the species as well as according to thebody position (prone, supine) and mechanicalproperties of the organs surrounding the kidney.In this study, we described the properties ofisolated kidneys. If, for example, the perirenal fatwould not have been removed, the results of thedrop shatter test would have been affected by theproperties of the fat (whose mechanical character-istics and thickness can vary considerably amongindividuals). This is why the other experimentalstudies mentioned above used porcine kidneyswithout perirenal fat (Bschleipfer et al., 2002;Snedeker et al., 2005) or even used isolated tissueblocks (Farshad et al., 1999). Description of the

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Table 2. The measured data P0L were tested for equality to the predicted values PL, and both Hypotheses H0A and H0Bwere rejected.

Sample n Rupture P0L (mm�1) PL (mm

�1) p

Kidney No. 1V1, V2, D1, D2 40 C 0.005970.0031 0.063770.0061 po0.001D1, D2 20 M 0.009070.0036 0.064370.0058 po0.001Ca1, Ca2 20 – – – –

Kidney no. 2V1, V2, D1, D2, Ca1, Ca2 60 C 0.004970.0058 0.066170.0044 po0.001V1 10 M 0.013570.0059 0.083170.0020 p ¼ 0.004

Kidney no. 3V1, V2, D1, D2, Ca2 50 C 0.013370.0211 0.082070.0068 po0.001D2 10 M 0.033870.0193 0.145570.0069 p ¼ 0.008Ca1 10 – – – –

P0L indicates the intensity of counted intersections of the rupture profile with the inner circumference of the renal tubules; PL indicatesthe theoretical prediction of the same value based on length density (LA) published in the Table 1; n indicates the number ofhistological sections grouped to one sample for the values of P0L, PL, and for the sign test; p indicates the p-value of the sign testcomparing the corresponding values of P0L and PL; V1-2, D1-2, and Ca1-2 indicate ventral, dorsal, and caudal location and the numberof the sample. Kidney No. 1–3 indicates the number of the kidney under study; C and M indicate cortical and medullar ruptures,respectively;-indicates ‘‘not found’’ or ‘‘not defined’’.

Table 3. Average values with standard deviations (SD) of volume fraction of tubules (%) in the cortex and in themedulla.

Kidney No. VV (tubules,cortex) (%) SD VV (tubules,medulla) (%) SD

1 90.9 3.7 49.6 4.12 91.9 3.2 54.7 3.93 88.5 4.4 52.8 3.7

Each value was estimated in 10 slices sampled from the 6 tissue blocks from each kidney. Kidney 1–3 indicates the number of the kidneyunder study.



boundary conditions of porcine kidneys wouldrequire a study similar to that performed in humans(Tonar et al., 2004; Snedeker et al., 2007).

Microscopic assessment of ruptures

A number of quantitative descriptions of the ratkidney parenchyma, including the number ofglomeruli, the average length of glomerular capil-laries, the average diameter of capillaries, and thenumber of capillaries per glomerulus have alreadybeen reported (Nyengaard, 1999). This comprehen-sive paper also reviewed most of the stereologicaltechniques applicable in kidney research, includingthe sampling strategy and variability assessment.To our knowledge, the only microscopic assessmentof traumatic changes in perfused porcine kidneyshas been reported by Back et al. (1994). However,this paper described the dose-dependent disinte-gration of tubular cells and disruption of reticularfibres in shockwave-induced lesions, which can notbe considered a model of blunt renal injury.

The assumption of isotropy of the profiles of therenal tubules has not been tested. Our study was atwo-dimensional assessment of rupture profiles; weassessed the damage to the tubule profiles andblood vessel profiles in micrographs rather than inthe three-dimensional real structures. The isotropyof the profiles of the tubules might have beenaffected by the local parallel spatial arrangementof the tubules. This might have also affected thepath of the ruptures, in particular, the rupturesstarting at the kidney surface. The section plane ofour samples was perpendicular to the kidneysurface, but randomly rotated around a verticalaxis perpendicular to the kidney capsule. Webelieve that it is impossible to assess the errorcaused by the local parallel arrangement of thetubules without a full three-dimensional study.

For assessment of LA, we chose the innercircumference of the tubules instead of the outercircumference. As we evaluated the inner circum-ference of the tubules, we also counted theincomplete ruptures of the tubular epitheliumwhere the inner circumference was disrupted andthe outer was not. The LA of the outer circumfer-ence would be larger than that LA of the innercircumference. As PL is directly proportional to LA,a PL value based on the length density of profiles inthe outer circumference would have been higherthan the PL value based on the length density ofinner circumference. However, the intensity ofintersections of the rupture profiles with the innercircumference of both cortical and medullartubules P0L would have been unchanged because

incomplete ruptures were extremely rare (thisoccurred only twice in all series and thus couldnot have affected the results). If we had assessedPL based on the length density of the outercircumference, we would have tested artificiallyincreased values against the lower P0L values, whichwould have weakened the results of the sign test.

We estimated only the maximal projection lengthof the cracks instead of estimating the length of therupture profiles. The rationale for this method wasthat the cracks were mechanically initiated by thestraight edges of the metal plate, and thismechanical triggering corresponded statisticallyto randomly placed lines. This measurementtechnique eventually underestimated the length l;thus, the rejection of the hypotheses was strength-ened (conservative decision).

The results demonstrate that microcracks of theparenchyma may occur even without macroscopicruptures. This is in agreement with the commonexperience of physicians treating the urogenitalsystem, as some patients with haematuria andanamnestic trauma have negative results on ultra-sonography of kidneys. It might be of interest totest whether the cracks beginning at the surfaceand the inner cracks have different P0L values. Suchdata could provide insight about crack behaviour atthe microscopic level. It would be especially usefulto determine whether the P0L values differedamong samples taken from ventral, dorsal, orcaudal parts of the kidney, or whether P0L valuesdiffered between the cortex and the medulla. Thehighest p-values for the sign test (Table 2) werefound in the samples of medulla (sample V1 ofKidney 2, sample D2 of Kidney 3), but this wasbiased by the lower number of slices collected forthese samples. Thus, we do not have enough datato publish a detailed analysis in this paper.

All the ruptures in the experimental kidneys(nos. 1–3) were followed in at least five consecutiveserial sections; thus, we are almost sure they wereof traumatic origin and not artefacts produced byhistological processing. However, for conclusiveproof, a 3-D reconstruction of the ruptures basedon thick sections and whole-block staining wouldhave to be performed; thus dubious ruptures wereexcluded from quantitative assessment to ensure avalid analysis. Ten tubules were tracked as far aspossible, and none were observable in a serieslonger than 45 mm. As the distance betweensections sampled for analysis was 100 mm, weconsidered the ruptured tubules counted in twoadjacent sections to be independent. Therefore,the only sectioning artefacts present in the experi-mental samples were occasional missing glomerulithat were not relevant to our study. It is certain

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that the autolysis of most kidneys had alreadystarted before they were fixed. However, webelieve it unlikely that the pressure of salineinjection could produce artefacts that might havebeen mistaken for ruptures. As ruptures found inone of the 18 tissue blocks sampled from thecontrol (perfused, but not dropped) kidneys couldnot be tracked in the third dimension in serialsections, we consider these ruptures to be section-ing artefacts rather than damage produced bymechanical manipulation. As the maximum projec-tion length values of the ruptures and the actualand hypothetic number of intersections with thetubules in Table 1 referred to the two sporadicmicrocracks found in the control samples, it wasimpossible to test the significance of the differencebetween P0L and the PL values with n ¼ 2 in thecontrol samples. It was therefore also impossible toassess the statistical significance of the higheroccurrence of the ruptures in the experimentalsamples (19 ruptures in 18 tissue blocks) whencompared to the controls (1 rupture in 18 tissueblocks). Although we evaluated only three kidneysfrom the 26 initially obtained, the low variability ofour results presented in Tables 1–3 suggest no needto increase the number of kidneys used forhistological examination.

Relation of the experiment to kidneymodelling and clinical medicine

It was expected that the tubular walls would be(within limits) more resistant to shear stress thanthe fine reticular connective tissue. Our resultsconfirmed that the cracks tended to run within theinterstitial connective tissue. A local parallelarrangement of the tubules may have strengthenedthis behaviour. The relevance for modelling is thatthe kidney parenchyma can be modelled as aporous medium consisting of tubular structuresthat are more cohesive to each other than to thesurrounding interstitial phase, which represents theconnective tissue. For this purpose, Cimrman andRohan (2007) used a neo-Hookean material withthree pressure systems (blood inflow, blood out-flow, and filtrate outflow) associated with parallelporosities (kidney tubules and blood vessels) inter-penetrating the material matrix and mutuallyseparated by interface sectors. Although descrip-tion of a realistic continuum model of living tissueremains one of the largest challenges for con-tinuum mechanics, the current scale-dependentapproach seems to be promising (Holecek andMoravec, 2006) as it incorporates quantitative

microscopic morphology into macroscopic modelsof organs and tissues.

The experiment performed in this study can becompared to grade I on the injury scale ofthe American Association for Surgery in Trauma(Moore et al., 1989). These injuries representapproximately 82% of kidney injuries (Harris etal., 2001) and consist of renal contusion, micro-scopic or gross haematuria, and subcapsular non-expanding haematoma. As such injuries are almostalways treated conservatively, there is little under-standing of the microscopic damage to the par-enchyma. Moreover, a significant number of suchinjuries are likely to escape diagnosis, and thedegree of haematuria does not correlate with thegrade of the injury (Brown et al., 2001; Ceylanet al., 2003). The source of haematuria has not yetbeen found, but might be the microcracks occurringin the cortex or in the medulla, microcracks hiddendeep within the parenchyma, or the rupture of thenephron and collecting ducts coincident with thedamage of blood vessels (as the pelvis and uretersare not affected in this grade of injury). At present,we are not aware of any published evidence frompathological materials that describes the relation-ship between patient history, clinical diagnosis ofgrade I or II kidney injury, and the presence of themicrocracks, as these injuries are not an indicationfor autopsy, and the patients usually recoverspontaneously. Therefore, the present study helpsto elucidate the mechanism of low-grade bluntinjury rather than facilitating its therapy, whichwill remain symptomatic and conservative inhaemodynamically stable patients, especially whenevidence-based algorithms for imaging methods areavailable (Blankenship et al., 2001; Harris et al.,2001). We have demonstrated the simultaneousrupture of arcuate vessels and adjacent corticaltubules (e.g., Figure 5B), which offers an explana-tion for gross haematuria. The rupture of smallcapillaries running through the cortical or medullarinterstitial connective tissue, which was demon-strated as the main pathway for the propagation ofthe rupture, might explain microscopic haematur-ia, as there is the potential for communicationbetween extravasated fluid and the lumen of thecracked tubuli.

Volume fraction of the tubules

We decided to include the volume fraction oftubules into this study for three reasons. Firstly, weconsider the volume fraction to be a suitableparameter somewhat complementary to the lengthdensity of the tubule profiles, which is the

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parameter upon which the analysis of the micro-cracks based. Secondly, it has been recentlysuggested (Cimrman and Rohan, 2007) that thereis a lack of morphometric data describing thetubular system of kidney; this lack of dataprevented the extension of the mathematicalmodel of kidney in that paper, which consideredthe kidney to consist of a porous material with acoupled fluid–structure interaction. The authors ofthat study were not aware of any relevantpublished computational study assessing the porouscharacter of the kidneys. Thirdly, we were sur-prised not to find any literature estimating thevolume fractions of cortical and medullar tubuleseither in human or in porcine kidneys. The onlyquantitative analysis recently published regardingthe volume of the cortical and medullar kidneytubules was performed in mice (Hoseiniet al., 2008) using the same stereological methodas has been applied in the present paper. Althoughthe previous results were more detailed (e.g., theparts of the nephron and the collecting ductswere assessed separately and the data also de-scribe the length of the tubules), the volumefraction of the cortical and medullar tubules werecomparable with our results. Surprisingly, Hoseiniet al. (2008) only used an overall azan stainingmethod, which makes the analysis of the medullapartly questionable. We are aware that neither theMallory stain used in our study nor the azan stainused by Hoseini et al. (2008) can guarantee reliablediscrimination between the thin segments ofHenle’s loops (which belong to the nephron) andthe vasa recta, which have a similar morphology.The thin segments of the Henle’s loops are linedwith epithelial cells, the nuclei of which tend tobulge into the lumen. The walls of the vasa rectaare usually thinner than those of the thin limbs, andthey have fewer endothelial cell nuclei, thus theprofiles of the nuclei appear further apart. How-ever, none of these differences are consistentacross all cross sections of the tubules andcapillaries, especially because some of the capil-laries in perfused kidneys do not even contain redblood cells. Therefore, we consider that thevolume fraction of the tubules within the medulla(Table 3) might be under- or over-estimated by asmall degree. We suggest that these data only beused for mathematical modelling, in which approx-imate figures are usually sufficient. We are awarethat comprehensive quantification of tubules wouldalso have required an analysis of the directionalityand distribution of tubules in various cortical andmedullar zones as well as separate assessment ofthe proximal and the distal tubules. We might, forexample, expect that the volume fraction of the

tubules would differ between superficial andjuxtamedullar cortical zones. The analysis of themedulla will require another study using advancedstaining methods (e.g. immunohistochemistry orlectin histochemistry) to distinguish blood vesselsfrom thin tubules. Another question remainingunanswered is whether the proximal or distaltubules within the cortex sustained more damage.

Identifying the mechanisms of injury is anessential step in prevention, diagnosis, and treat-ment. These mechanisms might be elucidated withthe use of physical impact tests and biomechanicalmodelling, preferably linked to clinical knowledge.Such results can be used for improvement of safetyprecautions to prevent blunt abdominal trauma.The results presented in this paper are intended tobe used for devising a non-trivial computer modelof kidney parenchyma that could be used forcomputer simulations of crashes. In papers thatspecifically analyse automobile accidents, pendu-lum impact, drop test, or sled test simulations havebeen performed (Ruan et al., 2005, 2006), and thekidney was modelled as a viscoelastic brickelement. Another sophisticated model of renaltrauma using the compartmentalised geometry ofthe human abdomen and kidneys has recently beenderived from the Visible Woman project andpublished by Snedeker et al. (2005, 2007). Thismodel involved detailed geometric and mechanicalproperties of kidneys and their relationships toother organs, and its predictive abilities havealready been validated in experiments. In ouropinion, in order to develop computer simulationsof crash tests in the future, it would be alsosuitable to combine the macroscopic morphology ofthe organ and its boundary conditions with adescription of the microstructure.

In conclusion, the interstitial collagenous con-nective tissue and arcuate veins showed lessresistance than glomeruli, cortical and medullartubules, and arcuate arteries during a drop shattertest simulating low-grade blunt injury in porcinekidneys. No disruptions of renal calyces were found.The volume fraction of the tubules was 90% incortex and 52.4% within the medulla. Despite thedifferences in syntopy and boundary conditions ofthe kidney in pig and man, porcine kidneys are themost suitable and widely used animal model forimpact tests simulating blunt kidney injury.

Acknowledgements

Supported by the Ministry of Education, Youthand Sports of the Czech Republic under Project nos.

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MSM4977751303 and MSM0021620819, as well as bythe Academy of Sciences of the Czech Republicunder Project no. A100110502.

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