Cellular Injury Associated with Renal Thrombotic Microangiopathy in Human Immunodeficiency Virus–Infected Macaques
Stephan Segerer*,
Frank Eitner,
Yan Cui*,
Kelly L. Hudkins* and
Charles E. Alpers*
*Department of Pathology, University of Washington, Seattle, Washington; and Division of Nephrology, University of Aachen, Aachen, Germany.
Address correspondence to Dr. Charles Alpers, Department of Pathology, University of Washington, Medical Center, Box 357470, 1959 NE Pacific Street, Seattle, WA 98195. Phone: 206-543-5616; Fax: 206-543-3644; E-mail: calp{at}u.washington.edu
ABSTRACT. Pigtailed macaques infected with a virulent humanimmunodeficiency virus-2 (HIV-2) strain develop renal thromboticmicroangiopathy (TMA), which morphologically resembles aspectsof human HIV-associated TMA. Apoptotic cell death of microvascularendothelial cells might be a pathogenetic clue to this disease.For defining further the pattern of cellular injury in thismodel, serial kidney sections of 58 macaques infected with HIV-2and 7 uninfected controls were studied by routine microscopy,terminal deoxynucleotidyl-transferase-mediated dUTP nick-endlabeling (TUNEL), 4',6-diamidino-2-phenylindole staining, andimmunohistochemistry for single-stranded DNA, p53, the Wilms’tumor suppressor gene-1 peptide product, caspase-3, and theproliferation marker Ki67. Selected cases were further evaluatedby in situ end labeling and transmission electron microscopy.Kidneys of 13 HIV-2–infected animals contained a patternof cellular injury, which was characterized by (1) nuclear swellingwith an ultrastructural morphology different from apoptoticnuclei, (2) sharply demarcated areas of renal cells with chromatinnicks (TUNEL positive) and single-stranded DNA, (3) absenceof an inflammatory or proliferative response, (4) upregulationof p53 and loss of at least one cellular differentiation marker(Wilms’ tumor suppressor gene-1), (5) a tight correlationwith the diagnosis of renal TMA, and (6) a contrast betweenprofound changes in the renal cellular morphology and the apparentlyunaffected clinical condition of the host. This pattern of injury,which shares some features of both apoptotic and oncotic necrosis,might be involved in the pathogenesis of HIV-associated renalTMA in this model.
Apoptotic cell death has been defined as a crucial event inorgan development, regulation of the immune system, and in avariety of tissue injuries, which brought renewed interest tothe nomenclature and characterization of cell death (1–5).Apoptosis is a controlled form of cell suicide, which is morphologicallycharacterized by nuclear condensation (pyknosis) and breakingup of the nucleus into membrane-bound fragments, karyorrhexis(6,7). Despite the precision achieved in defining some pathwaysof apoptotic cell death in well-controlled cell systems, ourability to recognize, define, and distinguish it from otherforms of cell injury and cell death in the setting of tissueinjury in vivo remains problematic (8–10). A pitfall ofmorphologic studies results from the rapid disappearance ofapoptotic cells via phagocytosis by professional and nonprofessionalphagocytes (9,11). This efficient process leads to a small numberof cells with an apoptotic appearance at a given time in tissueand therefore might result in underestimation of the contributionof apoptotic cell death (12). An even more fundamental problemarises in the attempt to clearly define apoptosis and distinguishit from other forms of cell death (13). Currently, the evaluationof apoptotic cell death in tissue sections relies on the combinationof techniques that demonstrate typical but nonspecific featuresof apoptotic cells (e.g., DNA strand breaks) with the morphologyby light and/or electron microscopy.
The association between human immunodeficiency virus (HIV) infectionand thrombotic microangiopathy (TMA) has been increasingly recognized(14). Endothelial cell apoptosis has been implicated in thepathogenesis of HIV-associated TMA in humans as plasma obtainedfrom patients with this disorder induces apoptosis, as wellas procoagulant properties in cultured endothelial cells (15–17).We demonstrated that infection of pigtailed macaques with avirulent HIV-2 strain resulted in renal TMA, defined by thrombiin arterioles and glomerular capillaries, in approximately 20%of the animals (18). The lesion closely resembles the morphologyseen in the human disease. In the present study, we evaluatedthe renal pathology using markers of DNA strand breaks, proliferation,renal cell phenotype, and ultrastructural appearance. Consistentwith previous reports in humans, we found an increased numberof terminal deoxynucleotidyl-transferase-mediated dUTP nick-endlabeling (TUNEL)-positive cells. However, as detailed below,the pattern of injury and the morphology were not consistentwith the classical description of apoptotic cell death.
Tissue Samples
Renal specimens of 65 pigtailed macaques were included in thestudy. Fifty-eight macaques were infected with HIV-2, and 7uninfected macaques served as normal controls. The mean timeof infection was 187 d, ranging from periods between 0.5 and1877 d. Only two animals were infected for longer than 2 yr.The histologic lesions resembling renal TMA in the studied animalshave previously been described in detail (18). See the Analysisof the Material section for the definitions used to describethe lesion in the present study.
Kidneys were fixed in 10% neutral buffered formalin or in methylCarnoys solution (60% methanol, 30% chloroform, 10% acetic acid)and routinely embedded in paraffin. From each formalin-fixedtissue block, serial sections were cut at 4 µm. Tissuefor electron microscopy was fixed in half-strength Karnovsky’ssolution (1% paraformaldehyde and 1.25% glutaraldehyde in 0.1M Na cacodylate buffer [pH 7.0]).
Detection of DNA Fragments, Single-Stranded DNA, and Condensed Nuclei
For the detection of DNA fragments by TUNEL methodology, weused the TdT-FragEL DNA Fragmentation Detection Kit (OncogeneResearch Products, Boston, MA). A second TUNEL kit, based onthe incorporation of a brominated nucleotide and the detectionby an anti–brominated nucleotide antibody, was used in15 cases (TACS·XL-DAB, Trevigen, Gaithersburg, MD). Insitu end labeling, based on the Klenow enzyme, was used in aseries of 31 specimens (KLENOW-FragEL DNA Fragmentation DetectionKit, Oncogene Research Products). The materials were used accordingto the instructions of the manufacturer with minor modifications.For the TdT-FragEL DNA Fragmentation Detection Kit, the protocolis described in brief. Deparaffinized and rehydrated slideswere rinsed in TBS (140 mM NaCl, 20 mM Tris [pH 7.6]) and incubatedin Proteinase K (20 µg/ml in 10 mM Tris [pH 8]; OncogeneResearch Products) for 20 min at room temperature. Endogenousperoxidases were blocked by a 5-min incubation in 3% H2O2 inmethanol and slides were incubated with equilibration bufferfor 30 min. Sixty microliters of TdT Labeling Reaction Mixturecontaining 3 µl of TdT was added for 60 min. Specimenswere incubated in Stop Solution for 5 min, followed by a rinsein TBS. Slides were incubated in blocking buffer for 5 min,followed by an incubation with the ABC Reagent (Vector, Burlingame,CA). Color development was performed applying diaminobenzidine(Sigma, St. Louis, MO) with Nickel enhancement. Slides werecounterstained with methyl green, dehydrated, and coverslipped.Negative controls were performed by omitting TdT, and positivecontrols were performed by digestion with DNase.
A monoclonal antibody F7-26 (Chemicon, International, Inc.,Temecula, CA) can be used to detect single-stranded DNA (ssDNA)after thermal denaturation (19,20). The monoclonal antibodyF7-26 was used according to the instructions of the manufacturer.A peroxidase-conjugated monoclonal rat anti-mouse IgM antibody(Zymed, San Francisco, CA) was used as secondary reagent. Thecolor reaction was performed as described above.
For the detection of condensed nuclei 4',6-diamidino-2-phenylindole(DAPI) staining was used. Slides from formalin-fixed and paraffin-embeddedtissue were deparaffinized and coverslipped with VectashieldMounting Medium with DAPI (Vector).
Immunohistochemistry
We previously described the immunohistochemical techniques usedin this study in detail (21–23). The specificities ofthe monoclonal mouse anti-p53 antibody (clone BP53-12; Sigma),the polyclonal rabbit anti–Wilms’ tumor suppressorgene-1 (WT-1; C-19; Santa Cruz Biotechnology, Inc., Santa Cruz,CA), and the monoclonal mouse anti-Ki67 antibody (MIB-1; AmacInc., Westbrook, ME) have all been previously shown by immunoblotting,and the application for immunohistochemistry has been publishedas well (24–28). The specificity of the polyclonal rabbitanti-active caspase-3 (Pharmingen, San Diego, CA) antibody hasbeen tested by immunoblotting by the company.
Analysis of the Material
A blinded observer evaluated 100 consecutive glomeruli for eachspecimen and staining technique. Glomeruli were categorizedas positive when more than one tenth of the glomerular cellswere involved. Glomeruli with <5 WT-1 positive cells werelabeled as having reduced WT-1 expression. Five or more p53-positivecells within a glomerulus was the definition of a p53-positiveglomerulus. Ki67-positive cells were counted per glomerulusand per cortical field (measuring 0.0625 mm2).
Apoptosis was quantified on DAPI stains. Bright round condensednuclei were counted as a feature of classical apoptosis in 30high-power fields at a magnification of x1000. The InStat program(Version 3.0 for Windows; Intuitive Software for Science, SanDiego, CA) was used to calculate the two-tail P value (Fisher’sexact test), the Spearman rank correlation, and the nonparametricKruskal-Wallis test for comparison of means.
HIV-2–Infected Macaques Develop Areas of TUNEL-Positive Cells in the Kidney
We studied renal specimens from a total of 58 HIV-2–infectedpigtailed macaques and 7 uninfected controls. We identifiedrenal TMA in 13 HIV-2–infected animals (18). In two additionalspecimens, both from HIV-2–infected macaques, the morphologywas suggestive of TMA, with focal thickening and double contoursof glomerular capillaries and endothelial swelling but withoutovert thrombi. These two cases were classified as indeterminate.Therefore, of the 58 infected macaques, 13 were classified ashaving TMA, 2 as indeterminate, and 43 as having no renal TMA.
Areas of TUNEL-positive cells were found in 13 of 65 renal specimens(20%) in a blinded evaluation. Two different patterns of TUNELpositivity were distinguishable (Figure 1). In 10 cases, thenuclei of all cell types within broad areas of tissue, whichappeared centered around arteries, were swollen and TUNEL positive(arterial pattern, Figure 1, A through D). Involved were cellsof the arterial walls, adjacent veins, adjacent tubular epithelium,and adjacent whole glomeruli (Figure 2, compared with normalin Figure 3). The arterial pattern was usually widespread, witha mean of 51 of 100 glomeruli involved (range, 21% to 72%; Figure 1, A and B).
Figure 1. Different patterns of TUNEL-positive areas in HIV-2–infected macaques with renal TMA. (A and B) Renal specimen from an HIV-2–infected macaque with TMA stained by silver methenamine and TUNEL. Note the widespread involvement of renal tissue (B) and the sharp demarcation of the process. The arterial pattern involves arteries (arrowhead), surrounding tubular epithelium and whole glomeruli (D). Note the TUNEL-negative glomerulus with normal nuclear features (right) adjacent to the involved glomerulus (left, C and D). (E and F) Renal specimen from an HIV-2–infected macaque stained by silver methenamine and TUNEL. Only small areas of the kidney, including larger veins and the adjacent tubular cells, are involved (arrow, venous pattern). The arrowhead marks an artery adjacent to the involved vein, which is TUNEL negative (E, F). Magnifications: x40 in A, B, E, and F; x400 in C and D.
Figure 2. Glomerulus with TUNEL-positive cells. Consecutive sections of a renal specimen from an HIV-2–infected macaque with TMA were stained with periodic acid-Schiff (PAS; A) and TUNEL (B), and immunohistochemistry for WT-1 (C), p53 (D), and ssDNA (E) were performed. Nuclei were swollen and pale on the PAS stain (A, arrows). All glomerular cells were TUNEL positive (B). WT-1 expression was completely absent (C). The nuclei of the glomerular cells were positive for p53 (D) and ssDNA (E). Magnification, x400.
Figure 3. Representative normal glomerular staining pattern. Consecutive sections of a renal specimen from an uninfected macaque were stained with PAS (A) and TUNEL (B), and immunohistochemistry for WT-1 (C), p53 (D), and ssDNA (E) were performed. The illustrated glomerulus shows WT-1 expression in the podocytes but no glomerular TUNEL-, p53-, or ssDNA-positive cells (compared with Figure 2). Magnification, x400.
Besides the widespread arterial pattern, three cases demonstratedsmall areas of TUNEL-positive cells involving large veins, theadjacent tubular epithelium, and rarely parts of adjacent glomeruli(venous pattern; Figure 1, E and F). Adjacent arteries showedno TUNEL-positive nuclei in these cases (Figure 1F). The borderbetween involved tissue with TUNEL-positive nuclei and adjacentnormal renal tissue was strikingly demarcated (i.e., the injurywas identifiable as a field defect; Figures 1, B and F, and 4).In selected cases, these results were confirmed with additionalend-labeling techniques, which are based on different enzymes,different incorporated nucleotides, and different detectionsystems. The distribution pattern was virtually identical withall techniques.
Figure 4. Changes in protein expression in TUNEL-positive areas. Consecutive sections of a renal specimen from a HIV-2–infected macaque with TMA were stained with PAS (A) and TUNEL (B), and immunohistochemistry for WT-1 (C), p53 (D), and ssDNA (E) were performed. This figure illustrates the involvement of three glomeruli and one adjacent normal glomerulus in the left lower quadrant (arrow). The three involved glomeruli show the typical features with nuclear swelling (A), strong TUNEL (B), p53 (D), and ssDNA positivity (E). WT-1 is absent in the involved glomeruli but is present in the normal glomerulus (C). Magnification, x400.
The described findings were substantiated by immunohistochemistryfor ssDNA, a technique that is not based on end labeling ofDNA strand breaks. Staining for ssDNA showed an arterial patternin all 10 cases, and 3 cases showed a venous distribution, 2of which demonstrated the same pattern in the TUNEL stain. Thecorrelation between the percentage of positive glomeruli forssDNA and for TUNEL was highly significant (r = 0.97, P <0.0001, n = 62).
In kidneys from uninfected control macaques, only a small numberof scattered positive nuclei in the interstitium between tubularcells, in tubular epithelium, in leukocytes within the lumenof arteries, and rarely within glomeruli were TUNEL positive.The number and distribution are consistent with apoptotic cellsin normal human kidneys as described previously (29).
Areas of TUNEL-Positive Cells Are Almost Exclusively Found in Specimens with Renal TMA
A striking finding was that the 13 cases with TUNEL-positiveareas included 10 cases with TMA and the 2 indeterminate cases.All specimens with the arterial pattern and two of three withthe perivenous pattern were from cases with TMA. The associationbetween TMA and cases with TUNEL-positive areas was highly significant(P < 0.0001). When the indeterminate cases were consideredas normal, the sensitivity of the presence of TUNEL-positiveareas for the diagnosis of renal TMA was 0.77, the was specificity0.94, the positive predictive value was 0.77, and the negativepredictive value was 0.94. When the indeterminate cases wereconsidered as abnormal, the sensitivity was 0.8, the specificitywas 0.98, the positive predictive value rose to 0.92, and thenegative predictive value was 0.94.
Ultrastructure of Nuclei in TUNEL-Positive Areas Is Different from Both Apoptotic and Oncotic Necrosis
The most prominent histologic change was severe swelling anda pale appearance of nuclei on silver stains (Figures 1, C and D, and 2).On DAPI stains, cells with condensed, bright nucleiwere very rare. We found no differences between the groups inthe number of nuclei with this classical feature of apoptosis(data not shown).
Using the feature of swollen, pale nuclei in glomeruli in ablinded evaluation, we found an excellent correlation betweenthe percentage of glomeruli with more than one tenth of theglomerular nuclei positive for TUNEL and nuclear alterationsdetected in periodic acid-Schiff (r = 0.88, P < 0.0001, n= 65) or silver stains (r = 0.97, P < 0.0001, n = 58). Thesharply demarcated areas of TUNEL positivity demonstrated nosurrounding inflammatory reaction.
By transmission electron microscopy, nuclei involved in TUNEL-positiveareas were swollen and contained two patterns of chromatin organization.The nuclear center had a speckled but homogeneous electron density(Figure 5A, compared with the normal nuclei in Figure 5B). Therest of the nuclear area was electron lucent and contained smallelectron-dense bodies that had the appearance of condensed chromatin(Figure 5A, insert). Therefore, the normal nuclear ultrastructurewith the electron-dense heterochromatin in the periphery wasno longer apparent (Figure 5B). Tubuli with normal nuclear featureswere found next to glomeruli with the described ultrastructuralfeatures (Figure 5, arrow). All involved cell types, i.e., glomerularcells, endothelial cells, myocytes of vessel walls, and tubularepithelium, demonstrated the same ultrastructural features (Figures 5 and 6).Venous endothelial cells with these nuclear featureswere sometimes detached from the basal membrane (Figure 6A).Effacement of podocyte foot processes was found in glomeruli,where podocytes showed these nuclear changes (Figure 5A).
Figure 5. Ultrastructural features of glomeruli from a specimen with TUNEL-positive areas. Electron microscopy of a specimen from an HIV-2–infected macaque with TMA. (A) All different cell types of the glomerulus show nuclear swelling and nuclear changes. Note the normal nuclear appearance of the adjacent tubular epithelial cells (arrow). (Insert) Nuclear ultrastructural features of the nucleus of an endothelial cell (arrowhead) at high magnification. It contains a speckled but homogeneous organization of the center, surrounded by small, round, electron-dense particles. (B) Conserved glomerulus with normal nuclear ultrastructure from the same specimen as illustrated in A. Magnifications: x800 in A; x10,000in A insert; x2400 in B.
Figure 6. Ultrastructural features of veins, arteries, and epithelium in TUNEL-positive areas. Specimens from HIV-2–infected macaques with TMA. (A) Detachment of an endothelial cell with the described nuclear ultrastructural features. (B) Vascular wall of a small artery. Myocytes of the vessel wall show the typical nuclear features (arrow). Note red blood cells in the arterial wall. No endothelial cells are apparent covering the elastica interna (arrowhead). (C) Tubular epithelial cells in TUNEL-positive areas sometimes appeared detached from the basal membrane. Magnifications: x2400 in A; x1600 in B and C.
Podocytes in TUNEL-Positive Areas Lose the Constitutive Expression of WT-1
WT-1 plays a crucial role during nephrogenesis; is constitutivelyexpressed in mature, differentiated podocytes; and accordinglyserves as a useful phenotypic marker of these cells (Figure 3C)(26,30). WT-1 was absent in glomeruli in TUNEL-positiveareas but was expressed in adjacent normal glomeruli (Figures 2C and 4C).Decreased glomerular WT-1 expression was found inall cases with the arterial pattern and in one case with theperivenous distribution of TUNEL-positive cells. The correlationbetween the percentage of glomeruli with reduced WT-1 expressionand TUNEL-positive glomeruli was highly significant (r = 0.84,P < 0.0001, n = 64).
p53 Is Expressed and No Proliferative or Inflammatory Response Is Present in TUNEL-Positive Areas
Glomerular cells in control specimens did not express p53 (Figure 3).Nuclear p53 expression was detected in all 10 cases withthe arterial pattern in the same distribution as the TUNEL-positivecells (Figure 4). Five cases without TUNEL-positive areas containedp53-positive cells with a normal nuclear morphology on replicatetissue sections. All five of these specimens were from HIV-2–infectedanimals.
Both HIV-2–infected and normal control specimens containeda low number of proliferating cells in tubular epithelium, inthe interstitium between tubules, and occasionally in glomeruli.A higher number of Ki67-expressing cells were found at sitesof focal leukocytic infiltration. Animals with TUNEL-positiveareas contained a significantly lower number of Ki67-positivecells per glomerulus compared with the group of HIV-2–infectedanimals without these areas (0.27 ± 0.09 versus 0.81± 0.13; P < 0.05; Figure 7). Specimens from HIV-2–infectedanimals showed a trend toward higher numbers of Ki67-positivecells, but the differences compared with normal animals didnot reach statistical significance (Figure 7). TUNEL-positiveareas were not associated with inflammatory infiltrates.
Figure 7. Expression of the proliferation marker Ki67. Mean number of Ki67-positive cells per glomerulus (A) and per high-power field (HPF; B) in uninfected macaques, macaques with HIV-2 infection, and macaques with HIV-2 infection and TUNEL-positive areas (Bars give SEM; *P < 0.05)
The available measurements of serum creatinine (0.67 mg/dl [n= 38] versus 0.64 mg/dl [n = 10]) and blood urea nitrogen (26.3mg/dl [n = 40] versus 28.5 mg/dl [n = 11]) did not differ betweencases without and with TUNEL-positive areas. None of the animalshad clinical signs of impaired renal function or systemic illness.The strain of HIV-2 used is pathogenic for an acquired immunodeficiencysyndrome–like syndrome in macaques at time points laterthan those used in the prospective serial sacrifice portionof the study. These manifestations, like the human disease thatthey model, may occur months to years after infection and arenot invariably present as some macaques have survived >3yr without clinical symptoms despite documented infections.
The pathogenesis of TMA in retrovirus-infected individuals isstill obscure. Plasma from patients with acute thrombotic thrombocytopenicpurpura, with and without HIV infection, has been shown to induceapoptosis in microvascular endothelial cells but not in endothelialcells of large-vessel origin (15). A differential inductionof apoptosis by plasma from patients with TMA in dermal, renal,and cerebral microvascular endothelium (i.e., sites typicallyaffected by TMA) but not in endothelial cells of pulmonary andhepatic origin (i.e., sites typically not affected by TMA) hasbeen reported (31). In addition, increased numbers of TUNEL-positivemicrovascular endothelial cells were described in spleens frompatients with TMA (32). Recent studies identified potentiallinks between endothelial cell apoptosis and initiation of aprocoagulant phenotype, which included decreased productionof prostaglandin I2, increased procoagulant surface plebs, increasedtissue factor activity, and decreased anticoagulant factors(thrombomodulin, heparan sulfate, tissue factor pathway inhibitor)(16,17,31). These reports prompted us to conduct the currentstudy on the pattern of cellular injury in HIV-2–infectedmacaques with renal TMA using ultrastructural analysis in combinationwith markers of DNA injury, cell proliferation, and cellularphenotype. Increasing evidence suggests that the classical modesof cell death, apoptosis, and oncosis (summarized in a simplifiedway in Table 1) represent only the extreme ends of a range ofmorphologic representations of cell death (12). We describea pattern of cellular injury that shares features of both classicalforms of cell death (Table 1) (13,33). Markers of DNA strandbreaks demonstrated a positive reaction in sharply demarcatedareas of renal tissue. This distribution pattern involving areasof different cells fits to an oncotic lesion rather than anapoptotic form of cell death, which usually can be detectedonly in a low number of scattered single cells in tissue sections(7,12,34). The absence of an inflammatory response, however,is in stark contrast with an oncotic lesion, in which the spillof cytoplasmic content leads to an inflammatory response (13,33,35).A principle morphologic feature that separates this lesion fromapoptosis is the nuclear swelling (Figure 1) (33). We couldnot detect active caspase 3 in the lesion. This finding is consistentwith either a nonapoptotic pathway or a stage of apoptosis inwhich caspase 3 is not activated.
Table 1. Comparison between the patern of injury in HIV-2–infected macaques with the features of apoptotic and oncotic necrosis
Are cells in TUNEL-positive areas doomed to die? The profoundmorphologic changes and the DNA nicks, indicated by the strongstaining by the TUNEL method, might suggest a lethal injury.By electron microscopy, the nuclei show two patterns of chromatinorganization, with a central region of high electron density,which is surrounded by an electron-lucent area filled with smallelectron-dense particles. This ultrastructural morphology isdistinguishable from both of the conventional, well-describedforms of cell death discussed previously. However, the contrastbetween the large areas of injury, the absence of clinical symptoms,and the absence of residual or progressive lesions suggest asublethal insult.
The described pattern of injury was tightly associated withthe morphologic diagnosis of TMA. The 10 cases with an arterialdistribution pattern of TUNEL-positive areas were from 8 animalswith TMA and the 2 indeterminate cases. None of the noninfectedcontrols demonstrated TUNEL-positive areas. TUNEL positivityof endothelial cells might provide a pathophysiologic clue forunderstanding the pathogenesis of TMA in this model. A morphologichint of a potential prothrombotic surface is the observationof endothelial cells detached from basement membranes and henceexposing extracellular matrices, which might then serve as procoagulantsites in affected vessels (36).
Studies on the appearance of endothelial cells in patients withthrombotic microangiopathies demonstrated swollen endothelialcells commonly separated from the basement membrane with ruffledendothelial surface and pseudopodal extensions (32,37,38). Othernonspecific findings were increased mitochondria, enlarged Golgielements, and numerous lysosomes (reviewed in Kwaan (38)). Inspleens from patients with thrombotic thrombocytopenic purpura,TUNEL-positive endothelial cells and hyperchromatic nuclei weredescribed (32). We found that endothelial cells were commonlydetached from the basement membrane and demonstrated the describednuclear features not consistent with classical apoptosis. Someof the endothelial features described in the literature areconsistent with apoptotic cell death, but further ultrastructuralstudies are needed to define clearly the pattern of endothelialinjury in humans with renal TMA.
The tumor-suppressor gene p53 accumulates and is activated byvarious forms of DNA damage and may cause either cell-cyclearrest or apoptosis (reviewed in May and May (39)). The increasedglomerular expression of p53 in TUNEL-positive areas in combinationwith decreased cell proliferation, illustrated by significantlydecreased numbers of glomerular Ki67-positive cells, is consistentwith a p53-induced cell-cycle arrest. The absence of WT-1 inTUNEL-positive glomeruli illustrates the profound phenotypicchange of cells involved in this type of injury. We have noreason to believe that this last change is functionally implicatedin the TMA injury, but decreased expression of WT-1 has beendescribed in humans with HIV-associated nephropathy (26).
Although the TUNEL method has been widely used both in animalstudies and on human tissue, the features described here inaggregate have previously not been recognized and are currentlyrestricted to retrovirus-infected primates. Additional studiesneed to address whether this pattern of injury is related toTMA in the context of HIV or to HIV infection itself or whetherthis might be a cellular reaction specific for the primate responseto retrovirus-associated renal TMA. The cellular reaction furthermorehas to be defined as a certain stage of cell death or a formof cell and tissue injury, which might potentially be reversible.Our limitation to fixed tissue excluded in vitro experimentsto answer these questions.
Acknowledgments
This work was supported by Grants HL63652 and RR00166 from theNational Institutes of Health and by a grant from the Else Kröner-Fresenius-Stiftung(Bad Homburg v. d. Höhe, Germany). We thank Min Wen forassistance with electron microscopy.
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Received for publication April 16, 2001.
Accepted for publication August 23, 2001.
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Abstract
Introduction
